Future cardiac event biomarkers CCL3, CCL5 and CCL18

ABSTRACT

The present invention relates to the identification of chemokine biomarkers predictive of future acute coronary syndromes including unstable angina pectoris (UAP). The present invention also identifies particular chemokines as potential therapeutic targets for intervention in cardiovascular diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application under 35 U.S.C.371 of PCT/IB2008/002771 filed on Sep. 10, 2008, which claims thebenefit of GB 0717637.3, filed on Sep. 10, 2007, the contents of each ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the identification of chemokinebiomarkers predictive of future acute coronary syndromes includingunstable angina pectoris (UAP). The present invention also identifiesparticular chemokines as potential therapeutic targets for interventionin cardiovascular diseases.

BACKGROUND OF THE INVENTION

Acute coronary syndromes, including unstable angina pectoris (UAP), areassociated with a high morbidity and mortality. In general, UAP resultsfrom erosion or rupture of a vulnerable atherosclerotic plaquesuperimposed by occlusive thrombus formation and distal ischemia¹.Atherosclerosis is increasingly regarded as a dyslipidemic disorder witha strong inflammatory character². These inflammatory processes are inpart orchestrated by chemokines, which participate in the inflammatoryprocess by mediating monocyte recruitment to sites of injury, vascularsmooth muscle cell proliferation, neo-vascularisation and plateletactivation³⁻⁵. Furthermore, chemokines appear to play a role in cardiacischemia as well. Indeed ischemia was reported to lead to inducedexpression of chemokines in the myocardium or in the circulation,translating in the recruitment of leukocyte subsets and progenitor cellsto the injury zone to contribute to the injury repair⁶. Given theirdiverse and deep impact in cardiovascular diseases, chemokines might notonly serve as biomarkers of atherosclerosis, plaque disruption orischemia, but also represent attractive therapeutic targets⁷.

Approximately 50 chemokines have thus far been characterized and variousare seen to be implicated in atherosclerosis and atherothrombosis⁵. Infact plasma levels of Regulated on Activation Normally T-cell Expressedand Secreted (RANTES or CCL5), Fractalkine (CX3CL1) and MonocyteChemotactic Protein 1 (MCP-1 or CCL2) have already been shown in variousstudies to be altered in UAP or myocardial infarction⁸⁻¹¹. Nevertheless,prospective data on chemokine plasma levels and/or chemokine receptorexpression by circulating leukocyte subsets in acute coronary syndromesare lacking. Moreover, the use of such chemokines as markers of futurecoronary events has hitherto not been explored.

SUMMARY OF THE INVENTION

The present invention is based on a study to assess the levels of 11chemokines in refractory unstable angina pectoris. The inventorsexamined baseline chemokine plasma patterns of a prospective cohort ofpatients with unstable angina pectoris by a high throughput multiplexassay, which allows simultaneous quantification of multiple chemokinesin one single plasma sample¹². For prospective analysis, differentiallyexpressed chemokines at baseline were analysed in follow-up samples byELISA. Furthermore, peripheral blood mononuclear cells (PBMCs) wereexamined for chemokine receptor expression.

In a first aspect the present invention provides the use of chemokineCCL3 and/or CCL18 and optionally CCL5 as a biomarker for theidentification of whether or not a test subject is at increased risk ofan acute cardiovascular syndrome or event.

CCL3 is also known as MIP-1α (macrophage inflammatory protein alpha) andLD78a/b; CCL18 is also known as PARC, DC-CK-1, AMAC-1 or MIP-4; andCCL-5 is also known as RANTES regulated on activation, normal Texpressed and excreted and SISd.

The cardiovascular syndrome or event may comprise coronary arterydisease, atherosclerosis, acute myocardial infarction, arteriosclerosis,unstable angina pectoris (UAP), embolism, deep vein thrombosis, stroke,congestive heart failure or arrhythmia.

Biomarker as used in the present invention means that the level of CCL3and/or CCL18 and optionally CCL5 as determined (e.g. detected and/orquantified) in tissue sample or a sample of a body fluid of anindividual is a predictive indicator for a future acute cardiovasculardisorder as such and/or for monitoring the status and/or progression ofa disorder. The detection of such biomarkers may also be used to monitortherapeutic regimes and/or clinical trials in order to detect whether ornot a particular treatment may be effective.

The aforementioned cytokines may be identified in a sample of body fluidfrom a subject, or in cells obtained from a subject, such as from anatheroscterotic plaque, for example. The sample of a body fluid of anindividual may be derived from blood, e.g. isolated mononuclear cells,or from a blood fraction, e.g. plasma or serum, especially plasma. Forthe purposes of the above aspects and embodiments, the subject may be ahuman or any other animal. In particular embodiments the subject isselected from the group consisting of human, non-human primate, equine,bovine, ovine, caprine, leporine, avian, feline or canine.

CC13, CC118 and/or CCL5 as used herein includes full-length protein, aprotein fragment, a mutated protein, derivatives and secreting(producing) cells, e.g. leukocytes and receptors therefor. Fragments,mutants and derivatives of said chemokines are such that the biomarkercharacteristic of the chemokines is retained.

The use of the aforementioned chemokines as biomarkers according to thepresent invention means one or more of said chemokines is determined insaid sample of an individual e.g. with detection means including thoseas conventional in the field of assays, e.g. immunoassays, chemokines isdetected by an immunoassay such as enzyme linked immunoassays (ELISAs);fluorescence based assays, such as dissociation enhanced lanthanidefluoroimmunoassay (DELFIA), radiometric assays, multiplex immunoassaysor cytrometric bead assays (CBA); sensors. Detection means of thepresent invention include e.g. a molecule which specifically recognizesthe particular chemokine, e.g. a molecule which is directly orindirectly detectable. Detection means of the present inventionpreferably comprise an antibody, including antibody derivatives orfragments thereof, e.g. an antibody which recognizes said chemokine(s)e.g. a label bearing chemokine recognising antibody.

The label may be one as conventional, e.g. biotin or an enzyme such asalkaline phosphatase (AP), horse radish peroxidase (HRP) or peroxidase(POD) or a fluorescent molecule, e.g. a fluorescent dye, such as e.g.fluorescein isothiocyanate. The label bearing molecule, e.g. the labelbearing antibody, may be detected according to methods as conventional,e.g. via fluorescence measurement or enzyme detection methods.

CCL3, CCL18 and/or CCL5 secreting cells in a sample of a body fluid ofan individual, e.g. blood, may be determined using conventional methodssuch as e.g. as described below. Cells may be purified, e.g. separatedby a density gradient, from the sample, e.g. blood, and the purifiedcells obtained are stained. Anti-CCL18/3/5 antibodies, e.g. fluorescencelabeled anti-CCL18/3/5 antibodies, may be added to the stained cellpreparation, optionally after stimulation of the cells, e.g. withinterleukin-4, and the level of CCL18/3/5 secretion by cells orCCL18/3/5-secreting cells determined or expression of the CCL18 and/or 3and optionally CCL5 receptor(s) determined.

Optionally, the CCL18 and/or CCL3 and optionally CCL5 comprised in thesample or the CCL18/3/5 recognising e.g. detectable, molecule comprisedin the detection means, may be immobilised on a solid phase. Anappropriate solid phase includes e.g. one as conventional, e.g. aplastic plate like a polystyrene or polyvinyl plate, especially amicrotiter plate. Also microbeads can be used as a solid phase, e.g.coated microbeads. The solid phase can be coated with a coating materialthe nature of which depends e.g. on the label comprised in the detectionmeans. The coating material should be able to bind to the label, e.g.the label may be biotin and the coating material includes streptavidin,e.g. covalently bound to the solid phase.

In another aspect the present invention provides a method for screeningand/or in vitro diagnosing whether or not an individual is at increasedrisk of an acute cardiovascular syndrome or disorder, which methodcomprises;

-   a) providing a sample of a body fluid of an individual;-   b) determining a level of CCL3, and/or CCL18 and optionally CCL5 in    the sample;-   c) comparing the level of CCL3 and/or CCL18 and optionally CCL5 as    determined in step b) with a reference level from a sample of a body    fluid of a healthy control individual; and-   d) screening and/or in vitro diagnosing whether the level of said    CCL3 and/or CCL18 and optionally CCL5 as determined in step b) is    significantly different from said reference level.

As mentioned above the appropriate chemokine receptor may also bedetermined, for example by FACS analysis using an antibody, preferablylabelled, that specifically recognises the receptor.

Determination of CCL3 and/or CCL18 and optionally CCL5 is carried out asdescribed above, e.g. by using a molecule which specifically recognisesthe biomarker, e.g. an antibody, an antibody derivative, an antibodyfragment, such as e.g. an anti CCL3 or CCL18 antibody, e.g. acommercially available CCL3 or CCL18 specific antibody, e.g. by animmunodiagnostic assay method.

In order to further enhance the sensitivity and/or selectivity of thediagnostic potential of the aforementioned chemokines as biomarkers, themethods described herein may be used in conjunction with assessment ofclinical symptoms and/or with the determination of the level of at leastone other biomarker in the subject, wherein the amount of the at leastone other biomarker may also be indicative of cardiovascular disease ora predisposition thereto. The further biomarker(s) may be selected fromother chemokines or cytokines and risk factors that have been indicatedas biomarkers of disease, such as CXCL 10 (IP-10), C-Reactive Protein,troponin 1, creatine kinase, creatine kinase MB, CD40L, HDL, ESR,platelet counts, sex, a cardiac index, myoglobin and/or interleukin-6(preferred embodiment) or any other more or less predictive biomarker ofcardiovascular disorders.

In another aspect the present invention provides a method for monitoringthe therapeutic efficacy of the treatment of an individual with asubstance which is expected to have an effect on reducing or curing anACS disorder or disease which method comprises determining the level ofCCL3 and/or CCL18 and optionally CCL5 or receptor(s) therefor in asample of a body fluid of said individual and comparing it to the levelof CCL3 and/or CCL18 and optionally CCL5 prior to administration of saidsubstance.

In a further aspect, the present invention provides a method formodulating a chemokine response in a vertebrate suffering or predisposedto suffering from an acute cardiovascular syndrome (ACS) comprising thestep of increasing or decreasing, or otherwise altering, the functionalactivity of CCL3 and/or CCL18.

The term “modulating a chemokine” in the context of the presentinvention also includes within its scope substantially preventing orpurposefully inducing in a vertebrate chemokine functional activity.

The term “preventing or purposefully inducing chemokine functionalactivity” in the context of the present invention includes within itsscope increasing or decreasing the expression and/or intracellular orextracellular distribution and/or activity of at least one chemokine asdescribed herein.

Increasing the expression may occur as a result of increasing mRNAexpression, or by increasing gene transcription using methods known tothose skilled in the art. Those skilled in the art will appreciate thatthere are many suitable methods to increase or decrease the expressionof a nucleic acid sequence encoding a chemokine as herein described.

One skilled in the art will appreciate that the expression or functionof one or more chemokines may be increased or decreased by increasing ordecreasing the levels of chemokine mRNA respectively by posttranscriptional modulation. For example, interfering RNA may be used asa method to decrease chemokine RNA levels.

Increasing or decreasing the intracellular distribution may occur as aresult of the addition of chemokine binding proteins to theintracellular environment. Alternatively, the intracellular distributionmay be increased, decreased or altered by the addition or removal ofsignal sequences and/or leader sequences to the chemokine. Techniquesused in such procedures will be familiar to those skilled in the art.

Increasing or decreasing the activity of the chemokines can be broughtabout by bringing the chemokines into contact with inhibitors ofchemokines, or activators of chemokines and/or chemokine bindingmolecules. Examples include antibody, antibody fragment or nanobody;genetically/chemically modified chemokine or chemokine portion withagonistic or antagonistic activity; synthetic small molecular entity; orany other (mammalian, viral or bacterial) protein with CCL3, CCL5 orCCL18 activity modifying capacity. The term “contact” in the context ofthe present invention means does not require a physical contact. Afunctional contact, that is where the presence of the inhibitor oractivator or chemokine binding protein affects the activity of thechemokine, is sufficient. This may occur when, for example, a thirdprotein mediates the interaction/contact between the chemokine bindingmolecule and the chemokine. That is, the interaction is indirect.

Suitable inhibitors and activators include but are not limited toinhibitors of chemokine receptors. One skilled in the art will be awaresuitable inhibitors or activators. In addition co-factors or chemokinebinding molecules may affect their activity. Examples include antibodiesand fragments thereof (for example Fab, F(Ab′)₂, Fv, disulphide linkedFv, scFv, diabody). It will be appreciated that this list is by no meansexhaustive.

In a further aspect there is provided a screen for identifying a CCL3and/or CCL18 modulator for potential use in treating ACS, such as UAP,the screen comprising the steps of:

providing in vitro a cell capable of expressing CCL3 and/or CCL18;

contacting a test modulator molecule with said cell;

subjecting said cell to conditions whereby the cell would normallyexpress CCL3 and/or CCL18 in the absence of the test molecule and;

detecting whether or not the test molecule has a modulatory effect onCCL3 and/or CCL18 activity.

The modulatory effect is preferably an inhibitory effect on CCL3 and/orCCL18 activity and may, for example, refer to expression levels of themRNA encoding said chemokines, or to protein levels observed. Suchlevels can easily be determined by the skilled addressee, usingtechniques well known to the skilled man and as described herein.Typically a control may be run along side, the control comprising a cellto which the test molecule has not been added, in order to obtain a CCL3and/or CCL18 reference level.

Convenient cells for use in such a method include leukocytes orneutrophils.

According to the above aspects of the invention, advantageously thefunctional activity of CCL3 and/or CCL18 is modulated using any one ofmore of the methods selected from the group consisting of: administeringa pharmaceutically effective amount of said chemokine/s to thevertebrate; administering a pharmaceutically effective amount of one ormore inhibitor/s of said chemokine(s) to the vertebrate; modulating thetranscription of said chemokine(s) in the vertebrate; modulating thetranslation of chemokine(s) in the vertebrate; modulating thepost-translational modification of chemokine(s) in the vertebrate andmodulating the intracellular or extracellular distribution of saidchemokine(s) in the vertebrate.

In a preferred embodiment of this aspect of the invention, thefunctional activity of said chemokine(s) is modulated by administering apharmaceutically effective amount of one or more inhibitor/s of saidchemokine(s) to the vertebrate. Advantageously, the one or morechemokine inhibitor/s are selected from the group consisting of:chemical chemokine inhibitors, anti-chemokine antibodies and dominantnegative mutants of those one or more chemokines described herein.

The functional activity of one or both chemokines may be modulated. Oneskilled in the art will appreciate that these chemokines may act inisolation or synergistically. In addition there may be functionalredundancy in the activity of chemokines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described by way of exampleand with reference to the figures which show:

FIG. 1: Plasma levels of CCL5 and CCL18 as determined by multiplex instabilised and refractory patients with unstable angina pectoris at t=0(A). ELISA was used for temporal patterning at t=0, t=2 and t=180 days.CCL5 levels dropped significantly at t=2 and were at the same level att=180 (B), while CCL18 levels remained elevated at t=2 and dropped backat t=180 (C). Soluble CD40L levels peaked at t=0 and were lowered at t=2and t=180 (D). CRP levels showed a peak at t=2, and lowered tosub-baseline values (t=0) at t=180 (E). Values represent mean±SEM,*P<0.05, **P<0.001 and N.S.=non-significant.

FIG. 2: Upper quartile plasma levels of CCL5 and CCL18 weresignificantly associated with the occurrence of refractory ischemicsymptoms in unstable angina pectoris, while sCD40L and CRP quartilelevels did not show any significant correlation (A). Upper quartilelevels of CCL5 at day 0 are predictive for the necessity of futurerevascularisation procedures (B), while upper quartile levels of CCL18were predictive for acute coronary syndromes or recurrent symptoms ofunstable angina pectoris within the next 18 months (C,D). *P=0.02,**P=0.01, ^(#)P<0.01 and N.S.=non-significant.

FIG. 3: Quantitative PCR analysis showed a markedly down-regulatedexpression of CCL5 and CCL18 in non-stimulated PBMCs of patients withischemic symptoms at t=0 compared to PBMCs at t=180 (A). In contrastwith chemokine receptor surface protein expression in PBMCs, mRNAexpression of the CCL5 and CCL18 receptors CCR1, CCR3, CCR4 and CCR5 wasalso approximately at least 2-fold down-regulated at baseline (B).Values represent mean±SEM, *P<0.05 and **P<0.001.

FIG. 4: Protein expression in PBMCs of CCR3 and CCR5 showed a clearup-regulation of both receptors in CD14⁺ cells (A,B) and CD3³⁰ cells(C,D) at baseline. Triple gating for CD14, CD3 and CCR3/5 revealed thesame trend, although CD14⁺ cells displayed more prominent up-regulationof CCR3 and CCR5 expression than CD3⁺ cells (E,F). Analysis of totalCCR3 and CCR5 surface expression in all PBMCs also showed a dramaticup-regulation of CCR3 and CCR5 expression, indicating that the increasein CCR3 and CCR5 expression is only partly caused by CD3⁺ and CD14⁺positive cells (G,H,I). *P<0.05, **P<0.01 and ^(#)P<0.001.

FIG. 5: Stimulation of PBMCs for 6 hours with rCCL5 and sCCL18 showed nosignificant differences in CCR1 (A), CCR4 (C) and CCR5 (D) mRNAexpression. CCR3 expression was markedly down-regulated afterstimulation with sCCL18, but not with rCCL5 (B). Values representmean±SEM, *P<0.01, N.S.=non-significant, rCCL5=recombinant CCL5,sCCL18=synthetic CCL18.

FIG. 6: Assessment of heterophilic interaction between CCL5 and CCL18 onPAGE (18%). Lanes 1 and 2 show reference mobility of rCCL5 (7,851 kDa)and sCCL18 (7,855 kDa), both chemokines showed a poor tendency to form15.6 kDa homodimers. Lanes 3 and 4 were loaded with mixtures of rCCL5and sCCL18 in a 1:1 and 1:5 ratio (weight:weight), at which dimers havebeen crosslinked by incubation for 30 min at RT with 25 mMparaformaldehyde. Note the slightly higher electrophoretic mobility andslightly more yellowish staining of CCL18 monomer and dimer. The extentof dimer formation was not altered after co-incubation and subsequentcrosslinking of CCL5 and CCL18, indicating that CCL5 and CCL18 areprobably not engaged in any significant heterophilic crossinteractioneven at supra-physiological concentrations. The total protein load perlane was constant (2 μg)(A).

FIG. 7: The PAGE analysis was corroborated by MALDI-TOF MS analysis:CCL5 and CCL18 (10 pmol/μl) gave mass peaks at approximately 7,860 Da(M⁺; theoretical mass of CCL5 and CCL18 7,851 and 7,855 Da,respectively), with only minor peaks at approximately 15,730 Da,illustrating the low tendency to form homodimers (M₂ ⁺) (A,B). MALDI-TOFmass spectrometry of CCL18 that had been pre-incubated with CCL5 at a1:1 and 1:5 w:w ratio (total concentration 10 pmol/μl) in 50 mMHEPES/0.1 mM EDTA with paraformaldehyde gave an essentially similarpattern and dimer formation was equally marginal at both ratios (C,D).

FIG. 8: Total levels of CD 14⁺ cells (monocytes and neutrophils) did notdiffer between t=0 and t=180, whereas CD3⁺ cells showed a small (11.8%),albeit significant decrease at baseline (A). At the mRNA level, anincrease of HNP-3+ neutrophils was observed, suggestive of enhancedpost-ischemic neutrophil release However, the CCR2:CX3CR1 expressionratio, a measure of monocyte subset profile, was not differentiallyregulated (B). Values represent mean±SEM, *P=0.01, **P<0.001 andN.S.=non-significant.

FIG. 9: Demonstrates that a transient exposure of mice to elevatedlevels of CCL18 in the circulation (as effected by repeatedadministration of recombinant CCL18 protein) will aggravate thedevelopment of atherosclerosis and thereby enhance the risk ofcardiovascular disease

FIG. 10: Demonstrates that atherosclerotic plaque development in theaortic sinus of hyperlimidemic (LDLr−/−) mice with a deficiency of CCL3is sharply reduced.

FIG. 11: Circulating CCL3 levels in APRAIS were comparable with thoseseen in patients from the MISSION! cohort (A). Temporal CCL3 monitoringclearly shows the transient increase of CCL3 during ischemia, sincelevels were significantly lowered at t=180 compared to t=0 (B). *P=0.03and **P<0.001.

FIG. 12: Upper quartile levels of CCL3 at baseline are predictive forthe occurrence of acute coronary syndromes during follow-up (A).Furthermore, upper quartile levels were also indicative of recurrentischemic symptoms during or directly after hospitalisation (B). *P=0.01and **P<0.01.

FIG. 13: Plasma levels of CCL3 (A), CCL5 (B) and CXCL8 (C) weresignificantly elevated in AMI patients (●) versus controls (∘), whereasCXCL10 (D) showed the opposite pattern. *P=0.025, **P=0.006, ***P=0.02and #P=0.004.

FIG. 14: Assessment of IL-6, CCL3 and CXCL10 levels in LAD ligated orsham operated mice. Cardiac ischemia induced significantly elevatedlevels of IL-6, and CCL3, (A,B). On the other hand, CXCL10 displayed aninversed pattern (C). *P=0.007, **P=0.02, and ***P=0.03.

FIG. 15: Ligated mice displayed a significant increase in the percentageof circulating T-cells with a concomitant enrichment in the CCR5⁺ andCXCR3⁺ subsets (A-C). The increase in circulating T-cells wasaccompanied by a decrease in CCR5⁺ splenic T-cells, whereas no effectson total or CXCR3⁺ splenic T-cells was apparent (D-F). *P=0.04,**P-0.02, ***P=0.04 and P=0.004.

FIG. 16 shows temporal profiling of CCL3 expression in collar inducedcarotid artery plaques showed increased CCL3 production 2 weeks aftercollar placement (A). Rapid and steady induction was observed for themacrophage marker CD68 (B), while CD36 induction was somewhat delayed(C). **p<0.01 compared to base line (t=0).

FIG. 17 shows CCL3 expression in macrophages is strongly upregulatedupon LPS (50 ng/ml) (A) but not ox-LDL (10 ug/ml) (B) stimulation. LPSinduced CCL3 response in vivo is ablated in CCL3−/− chimeras (C, blackbars)) ***p<0.001.

FIG. 18 shows atherosclerotic lesions were significantly smaller inCCL3^(−/−) chimeras compared to WT controls (A with representativepictures). Macrophages (B), collagen (C) and T cell content (D) wassimilar between WT and CCL3^(−/−) chimeras. Neutrophil influx (E) andadhesion (F) was significantly attenuated in CCL3^(−/−) chimeras. Blackbars represent WT controls and white bars CCL3^(−/−) chimeras. *p<0.05,**p<0.01.

FIG. 19 shows total number of white blood cells (A) and monocytes (B)was not different in CCL3−/− mice, whereas neutrophil numbers (C) weresignificantly decreased. Black bars represent WT and white barsCCL3^(−/−) chimeras. **p<0.01, ***p<0.001.

FIG. 20 shows kinetics of cyclophosphamide induced transient leukopenia(A) and neutropenia (B) in control (white bars) and CCL3^(−/−) mice(black bars). Elimination of neutrophils is accelerated in in CCL3^(−/−)chimeras (C), while repopulation is similar (D). Black bars represent WTmice and white bars represent CCL3^(−/−) mice. *p<0.05. **p<0.01.

FIG. 21 shows intra peritoneal injection of KC did not affectcirculating CD11b⁺ CD71^(− Gr)1^(high) neutrophil numbers in WT andCCL3^(−/−) mice (A). KC elicited induction of neutrophil influx to theperitoneal cavity was ≈2.5 times lower in CCL3^(−/−) mice compared to WTmice (B). Black bars represent WT mice and white bars representCCL3^(−/−) mice. **p=0.003,

EXAMPLE 1 CCL5 (RANTES) and CCL18 (PARC) are specific markers ofrefractory unstable angina pectoris and are transiently raised duringsevere ischemic symptoms.

Methods

Study Population

All chemokines and inflammatory parameters were determined in plasmasamples of a patient cohort, derived from the well defined APRAIS (AcutePhase Reaction and Ischemic Syndromes) study¹³. In brief, 54 patientswho were admitted to the emergency department of the Leiden UniversityMedical Center between March and September 1995 with unstable anginapectoris Braunwald class IIIB were included and followed for up to 18months. Venous blood samples were obtained on admission (t=0) after 2(t=2) and 180 days after admission (t=180), centrifuged and plasmaaliquots were stored at −80° C. until further analysis. All patients hadreceived standard medical therapy, i.e. aspirin 300 mg orally,nitro-glycerine intravenously and heparin infusion based titrated to theactivated partial thromboplastin time. A clinical end point of theAPRAIS study was the occurrence of refractory unstable angina pectorisduring hospitalisation. Unstable angina pectoris was consideredrefractory if angina at rest, despite medical treatment, remained orre-occurred, prompting invasive coronary assessment and subsequentrevascularization therapy. Although the study cohort was relativelysmall, it constituted a clearly defined, well documented population witha similar starting point. All subjects gave written informed consent andthe study protocol was approved by the Ethics Committee of the LeidenUniversity Medical Center.

Isolation of Cells

PBMCs from patients (t=0 and t=180) as well as from 6 healthy agematched volunteers were isolated from venous EDTA blood samples throughdensity centrifugation on Histopaque (Sigma, St. Louis, Mo.). PBMCs werecollected from the interphase and washed twice with culture medium,consisting of Iscove's modified Dulbecco's medium containing glutamax(Gibco, Paisly, UK) and supplemented with 10% FCS. PBMCs werecryopreserved in culture medium containing 20% FCS and 10%dimethylsulfoxide until further use.

Multiplex Chemokine Assay

Circulating levels of the chemokines CCL2, CCL3, CCL5, CCL11, CCL17,CCL18, CCL22, CXCL8, CXCL9, CXCL10 and the chemokine like factor MIF,the cytokines OSM, IFN-γ and OPG and adhesion molecules sRank1, sVCAMand sICAM were determined in t=0 samples with a custom made multiplexbio-assay using the Bio-Plex Suspension Array system (Bio-Radlaboratories, Hercules, Calif.) Plasma samples were filtered andsubsequently diluted with 10% normal rat and mouse serum (Rockland,Gilvertsville, Pa.) to block residual non-specific antibody binding.1000 microspheres were added per chemokine (10 μl/well) in a totalvolume of 60 μl, together with standard and blank samples, and thesuspension incubated for 1 hour in a 96 well filter plate at roomtemperature (RT). Then, 10 μl of biotinylated antibody mix (16.5 μg/ml)was added and incubated for 1 hour at RT. After washing with PBS-1%BSA-0.5% Tween 20, beads were incubated with 50 ng/well streptavidinR-phycoerythrin (BD Biosciences, San Diego, Calif.) for 10 minutes.Finally, beads were washed again with PBS-1% BSA-0.5% Tween 20, and thefluorescence intensity was measured in a final volume of 100 μlhigh-performance ELISA buffer (Sanquin, Amsterdam, the Netherlands).Measurements and data analysis were performed with the Bio-PlexSuspension Array system in combination with the Bio-Plex Managersoftware version 3.0 (Bio-Rad laboratories, Hercules, Calif.) (see alsoref. No: 14)

ELISA and Other Assays

For temporal analysis of human CCL5 and CCL18 plasma levels duringfollow up, the t=0, t=2 and t=180 samples were assayed by a CCL5 instantELISA kit (Bender MedSystems, Vienna, Austria) and a CCL18 ELISA(RayBiotech, Norcross, Ga.), respectively, according to manufacturersprotocol. Baseline inflammatory parameters such as C-reactive protein,fibrinogen, erythrocyte sedimentation rate (ESR) and plasminogenactivator inhibitor 1 (PAI-1) were determined as described in detailpreviously¹³. Soluble CD40 ligand (sCD40L) and Interleukin 6 (IL-6) weredetermined via a highly sensitive immunoassay (Quantakine HS, R&DSystems, Minneapolis, Minn.), t=180 CRP samples via a turbidimetricassay on a fully automated Modular P800 unit (Roche, Almere, theNetherlands).

Assessment of Heterophilic CCL5 and CCL18 Interaction

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) wasused to assess whether recombinant CCL5 (7.8 kDa) and synthetic CCL18(7.8 kDa) engage in heterophilic interactions. Proteins (rCCL5, sCC118,rCCL5/sCCL18 at a 1:1 and a 1:5 weight ratio (w:w); 2 μg total proteinper lane) were incubated for one hour at RT in 50 mM HEPES/0.1 mM EDTAbuffer (pH=7.4), after which 25 mM of paraformaldehyde was added tocross link any formed homo- or heterodimers. After 30 minutes, proteinmixtures were denatured in loading buffer and subjected to SDS-PAGE(18%; 2 μg protein per lane, one hour at 70 mV and 30 minutes at 150mV), proteins were visualized by silver staining. Protein mixtures werealso analysed on a Voyager-DE Pro MALDI-TOF mass spectrometer(PerSeptive Biosystems, Framingham, Mass.).

RT-PCR Analyses

To assess expression of CCL5, CCL18, CCR1, CCR2, CCR3, CCR4, CCR5,CX3CR1 and human neutrophil peptide-3 (HNP-3) in PBMCs, mRNA wasisolated and analyzed. Guanidium thiocyanate-phenol was used to extracttotal RNA from PBMCs, samples were subjected to DNAse I treatment(Promega, Madison, Wis.) after which cDNA was generated using RevertAidM-MuLV reverse transcriptase (Fermentas, Burlington, Canada) accordingto manufacturer's protocol². Semi quantitative gene expression wasperformed using the SYBR-Green method (Eurogentec, Liege, Belgium) on anABI PRISM 7700 machine (Applied Biosystems, Foster City, Calif.) withprimers for CCL5, CCL18, CCR1, CCR2, CCR3, CCR4, CCR5, CX3CR1, CD11b andhuman neutrophil peptide-3 (HNP-3). Cyclophilin and Hypoxanthine GuaninePhosphoribosyl Transferase (HPRT) were used as housekeeping genes (seeTable 1 for primer sequences).

TABLE 1 Primer sequences used for RT-PCR analysis. Gene name ForwardReverse HPRT GAAATGTCAGTTGCTGCA ACAATCCGCCCAAAGGGAAC TTCCT Cyclo-AGTCTTGGCAGTGCAGAT GAAGATGAGAACTTCATCCTAAA philin GAA GCATA CCR1TCCTGCTGACGATTGACA GTGCCCGCAAGGCAAAC GGTA CCR2 TTCGGCCTGAGTAACTGTTGAGTCATCCCAAGAGTCTCTGT GAAA C CCR3 CTGCTGCATGAACCCGGTGGAAGAAGTGGCGAGGTACT CCR4 ACTGTGGGCTCCTCCAAA TCCATGGTGGACTGCGTG TTT CCR5AGACATCCGTTCCCCTAC CAGGGCTCCGATGTATAATAATT AAGAA GA CX3CR1GTCCACGTTGATTTCTCCT CGTGTGGTAAGTAAAATTGCTGC CATC T HNP-3CCCAGAAGTGGTTGTTTC TTTCCTTGAGCCTGGATGCT CCT CCL5 TCTGCGCTCCTGCATCTGCAGTGGGCGGGCAATG CCL18 CCTGGAGGCCACCTCTTC TGCAGCTCAACAATAGAAATCAA TAA TTRelative gene expression was calculated by subtracting the thresholdcycle number (Ct) of the target gene from the average Ct of Cyclophilineand HPRT and raising two to the power of this difference.Flow Cytometry

CCR3 and CCR5 surface expression on CD3⁺ and CD 14⁺ PBMCs was assessedby flow cytometry. Cryopreserved PBMCs were thawed, washed three timesin RPMI 1640 containing 20% FCS and subsequently stained using APCconjugated anti-CD3 and anti-CD14 antibodies (BD Biosciences, San Jose,Calif.) as well as FITC conjugated anti-CCR3 and anti-CCR5 antibodies(R&D Systems). Non-specific isotypes FITC conjugated Rat IgG2a and FITCconjugated mouse IgG2b antibodies (eBiosciences, San Diego, Calif.) wereused as negative controls. Samples were analysed with a fluorescenceactivated flow cytometer (FACSCalibur) and subsequently analyzed usingCELLQuest software (BD Biosciences), 50,000 cells were counted for eachsample.

PBMC Stimulation Assay

Cryopreserved PBMC specimens, obtained from six healthy volunteers werethawed as described above, plated in a U-shaped round bottom 96-wellplate (Greiner Bio-one) and stimulated for 6 hours at 37° C. with plainmedium (control) or medium supplemented with 50 ng/ml recombinant CCL5(Peprotech, Rocky Hill, N.J.), 50 ng/ml of the synthetic CCL18 peptideSM-1 (sCCL18), or a combination of rCCL5 and sCCL18 (25 ng/ml perpeptide)³. After incubation, total RNA was isolated from the cells, cDNAwas prepared and chemokine receptor expression was determined.

Statistical Analysis

Differences between our study populations and the original cohort wereexamined by Fisher's exact test and Student's unpaired t-test. Plasmalevels of chemokines and inflammatory markers were tested for normalGaussian distribution and values were log-transformed in the case of askewed distribution when appropriate. Regarding the latter, geometricinstead of arithmetic means are given. Means were compared by unpairedtwo-tailed Student's t-test or Mann-Whitney U-test when appropriate. Inorder to assess the predictive value of CCL5 and CCL18 for theoccurrence of refractory symptoms, independent of potentiallyconfounding factors, a multivariate analysis was performed, correctingfor age, HDL and ESR levels, as well as for other establishedcardiovascular risk factors (e.g. hypertension, hypercholesterolemia,use of lipid and blood pressure lowering medication, diabetes mellitus,smoking behaviour, BMI and history of cardiovascular disease) andbiomarkers sCD40L and CRP. Quartile distribution was assessed and usedfor Spearman's correlation coefficient and Pearson's chi-square testingto determine the association of chemokine plasma levels as well aslevels of sCD40L and CRP for the occurrence of refractory UAP. Receiveroperating characteristics curves were generated to assess predictivevalue of chemokines for refractory ischemic symptoms. Correlationanalysis between multiplex and ELISA values and between chemokines andinflammatory parameters were performed by Spearman's rank correlationtest. FACS results were analysed via paired t-test, the stimulationassay was analysed via ANOVA. A two-sided p-value<0.05 was consideredsignificant. All analyses were performed using SPSS version 14.0software (SPSS, Chicago, Ill.).

Results

Study Population

Plasma analyses on chemokines were performed in a subcohort ofpreviously unfrozen plasma samples of 54 consecutive patients, excludingselection bias. This subcohort, consisting of 31 patients withstabilised and 23 with refractory ischemic symptoms, matched with theoriginal cohort on cardiovascular risk factors, history of myocardialinfarction or PTCA/CABG and laboratory parameters (Tables 2A and B).

TABLE 2A Baseline patient characteristics and laboratory parameters.Chemokine cohort APRAIS (N = 54) (N = 211) P-value Age, years  65.4 ±11.0 62.7 ± 10.2 0.08 Refractory (%) 43 36 0.43 Male gender (%) 73.871.1 0.75 Current smoker (%) 24.6 30.5 0.45 BMI (kg/m²) 25.2 ± 6.0 25.9± 3.36 0.23 Diabetes (%) 16.4 14.6 0.98 Hypertension (%) 23 23.5 0.99Statin use (%) 8.2 12.2 0.48 History of: Myocardial infarction 45 43.20.88 (%) PTCA (%) 26 29.1 0.75 CABG (%) 23 21.6 0.86 Laboratoryparameters: Total cholesterol, 6.00 ± 1.5 6.18 ± 1.2  0.38 mmol/l HDL,mmol/l 1.14 ± 0.4 1.14 ± 0.3  0.97 CRP, mg/l * 2.36 2.66 0.50 ESR,mm/hr * 16.44 14.88 0.30 Fibrinogen, g/l * 3.56 3.42 0.34

TABLE 2B Chemokine cohort baseline patient characteristics andlaboratory parameters Stabilised Refractory (N = 31) (N = 23) P-valueAge, years  67.3 ± 10.2  64.5 ± 11.4 0.30 Male gender (%) 87 63 0.05Current smoker (%) 22 25 0.69 BMI (kg/m²) 24.9 26.7 0.78 Diabetes (%) 919 0.16 Hypertension (%) 17 38 0.39 History of: Myocardial infarction 4847 0.89 (%) PTCA (%) 30 25 0.57 CABG (%) 35 16 0.19 Laboratoryparameters: Hemoglobine, mmol/l 8.27 ± 2.1 8.51 ± 0.8 0.61 Hematocrite(%) 47 41 0.26 Leucocytes, 10⁹/l 7.49 ± 2.9 7.68 ± 2.2 0.79 Plateletcount, 10⁶/l 186.5 ± 66   223.9 ± 75   0.07 Glucose, mmol/l 7.37 ± 2.76.49 ± 1.4 0.15 Creatinine, μmol/l  99.2 ± 52.3 108.7 ± 32.1 0.44Cholesterol, mmol/l 5.92 ± 1.8 6.16 ± 1.0 0.56 HDL, mmol/l 1.23 ± 0.40.99 ± 0.2 0.02 ESR, mm/hr * 14.15 20.70 0.03 Fibrinogen, g/l * 3.423.78 0.26 CRP, mg/l * 2.14 2.77 0.47 sCD40L, pg/ml 23.6 20.3 0.32

As not all 54 patients responded to donate blood after 180 days, ELISAanalysis at this point was performed for 47 patients (stabilised 29 vs.refractory 18), but the baseline characteristics of this subcohortmatched with that of the original cohort (data not shown). Comparisonfor baseline demographics in the chemokine cohort showed no strikingdifferences between refractory versus stabilised patients, except for asmall, but significant difference in gender composition (87% vs. 67%males; P=0.05); the mean age of all patients was 65 years (41 to 85years). Regarding the clinical and plasma lipid parameters at baseline,total cholesterol levels in stabilized and refractory patients did notdiffer (5.92 vs. 6.16 mmol/l; P=0.56), whereas HDL levels were lower(1.23 vs. 0.99 mmol/l; P=0.02) in the latter population. This group alsodisplayed an increased tendency towards a higher inflammatory status, asillustrated by elevated levels of the ESR (14.15 vs. 20.7 mm/hr; P=0.03)albeit that fibrinogen and CRP levels were essentially similar. Nodifferences were observed in baseline sCD40L levels between groups.

Multiplex Analysis: Upregulation of CCL5 and CCL18

All of the chemokine and cytokine data as determined by multiplexanalysis (t=0 samples) were log-transformed before further statisticalanalysis because of their skewed distribution profiles, except for OPG.Plasma levels of the majority of chemokines and cytokines did not differbetween stabilized and refractory patients. CCL5 (23.1 vs. 32.7 ng/ml;P=0.018) and CCL18 levels (53.7 vs. 104.4 ng/ml; P=0.011) howeverappeared to be significantly increased in refractory patients, whilethere was a borderline significant increase in those of CCL3 (53.6 vs.73.7 pg/ml; P=0.09) (Table 3 and FIG. 1A).

TABLE 3 Chemokine plasma concentrations analysed via the multiplextechnique. Variable Stabilised Refractory P-value CCL5, pg/ml 2315832704 0.018 CCL18, pg/ml 53678 104399 0.011 CCL2, pg/ml 154 146 0.77CCL3, pg/ml 53.6 73.7 0.09 CCL11, pg/ml 63.7 65.8 0.88 CCL17, pg/ml 40.351.2 0.34 CCL22, pg/ml 527 546 0.79 CXCL8, pg/ml 12.4 13.4 0.84 CXCL9,pg/ml 158 156 0.96 CXCL10, pg/ml 221 157 0.12 MIF, pg/ml 330 439 0.45OPG, pg/ml* 937 1096 0.25 OSM, pg/ml 456 690 0.25 sRankL, pg/ml 5.0 5.70.83 sVCAM , pg/ml 681082 735190 0.45 sICAM, pg/ml 106340 117625 0.28Values are geometric means *denotes arithmetic meanMoreover, the observed differences in CCL5 levels remained significantafter multivariate analysis adjusting for cardiovascular risk factorsand sCD40L and CRP levels (P=0.023), whereas CCL18 levels wereborderline significant (P=0.06). However, differences in CCL 18 levelsreached significance after multivariate analysis for all confoundingfactors but HDL (P=0.021). Therefore, CCL5 as well as CCL18 seem to beindependent predictors of the occurrence of refractory ischemicsymptoms, even when adjusting for sCD40L and CRP levels. Furthermore,CCL5 and CCL18 levels showed no mutual correlation (R=0.05; P=0.7),reflecting that these chemokines are regulated or operate in anindependent manner. Still, although no significant heterophilicinteractions between CCL5 and CCL18 were observed, it is conceivablethat both chemokines, sharing CCR3 as common target receptor willinteract functionally (FIGS. 6 and 7). CXCL10 had a tendency to rise instabilised patients, although not quite significant (221.6 vs. 157.5pg/ml; P=0.12), which could point towards a protective effect of thisspecific chemokine. Levels of IFN-γ were merely undetectable and aretherefore not shown.

Next, we sought to assess if CCL5 and CCL18 levels have diagnosticpotential. Given the cohort size, levels of CCL5 and CCL18 werecategorized into quartiles and analyzed for correlation with theoccurrence of future refractory ischemic symptoms (Table 4A).

TABLE 4A CCL5 and CCL18 quartile levels at baseline as determined bymultiplex analysis Quartiles CCL5 CCL18 1 <15.1  <39.3 2 >15.1 and<25.5 >39.3 and <66.0  3 >25.5 and <40.3 >66.0 and <130.0 4 >40.3 >130.0All values are in ng/ml

The risk of refractory ischemic symptoms was seen to be increased in theupper quartiles of CCL5 (R=0.32; P=0.017; Linear-by-linear associationchi-square 5.53; P=0.019), while this trend was even more pronounced forCCL18 (R=0.392; P=0.003; linear-by-linear association chi-square 8.105;P=0.004) (FIG. 2A). Elevated CCL18 levels were slightly more predictivethan those of CCL5 as indicated by the receiver operatingcharacteristics curve (area under the curve 0.71 vs. 0.69). Cut-offvalues of >40 ng/ml for CCL5 and >130 ng/ml for CCL18 yielded asensitivity of 73.9% and 65.2%, respectively as well as a specificity of67.7% and 61.3%. Combined analysis of the upper two quartiles of CCL5and CCL18 for the occurrence of refractory ischemic symptoms revealed avery significant relation (χ² with continuity correction 8.12; P<0.01).While the sensitivity reached 47.8%, the specificity of the combinedanalysis was a remarkably high 90.3%. The positive predictive value ofcombined analysis for CCL5 and CCL18 levels was 78.5% with a concomitantnegative predictive value of 70.0%. Adding sCD40L or CRP levels to theanalysis did not yield any further increase in sensitivity, specificityor predictive value (data not shown).

CCL5 and CCL18 ELISA Verification and Follow-Up Analysis

Mean and individual ELISA and multiplex CCL5 levels correspondedexcellently (P<0.001). Moreover, CCL5 plasma levels were also seen to beincreased in refractory compared to stabilised patients at day 0 whenassessed by ELISA (36.4 vs. 26.5 ng/ml). Interestingly, already aftertwo days, a marked decrease in plasma CCL5 levels was observed in thewhole cohort (12.1 versus 30.3 ng/ml; P<0.001) and reduced CCL5 levelswere also observed at t=180, showing that CCL5 is transiently raisedduring an episode of unstable angina pectoris (FIG. 1B). We did notobserve any differences between the stabilized and refractory groups at2 and 180 days post inclusion. Plasma levels of CCL18 showed a differenttemporal pattern after ischemic symptoms. ELISA analysis confirmed thedifferential expression of CCL18 at day 0 between refractory andstabilised patients (56.2 vs. 41.1 ng/ml; P=0.02). Although absolutevalues were slightly lower in the ELISA compared to the multiplex assay,statistical analysis revealed an excellent correlation between the twoassays (Spearman's test; P<0.001). Interestingly, CCL18 levels of thetotal cohort at day 2 did not differ with the baseline levels (day 0),suggesting that CCL18 and CCL5 levels might be regulated via separatemechanisms. At 180 days, CCL18 levels were significantly down-regulatedcompared the day 2 values (48.4 vs. 34.5 ng/ml; P<0.001), suggestive ofa role of CCL18 in cardiac ischemia-reperfusion related processes (FIG.1C).

Soluble CD40 Ligand and CRP

Levels of both sCD40L as well as CRP were significantly elevated at t=0compared to t=180 (sCD40L 2.04 vs. 0.69 ng/ml; P<0.001, CRP 2.36 vs.0.96 mg/l; P<0.001) (FIG. 1D,E). However, sCD40L levels started todecline already at t=2 (1.35 ng/ml; P<0.05) indicating that elevatedlevels at baseline reflect a platelet activation related acute phaseresponse. As soluble CD40L t=0 and t=2 levels at correlatedsignificantly with CCL5 t=0 and t=2 levels (t=0 R=0.40; P<0.01, t=2R=0.35; P=0.01), elevated CCL5 levels may be primarily caused byplatelet activation as well. sCD40L however showed a significantnegative correlation with CCL18 levels at t=0 (R=−0.36; P=0.01),suggesting that latter represent a feedback response to plateletactivation. CRP levels were even further increased at t=2 (6.43 mg/l;P<0.001) which is in keeping with previous reports^(15,16), andpresumably indicative of an enhanced post-ischemic systemic inflammatorystatus in these patients two days after ischemia and/or coronaryintervention. CRP levels showed no correlations with CCL5 or CCL18levels. Quartile levels of sCD40L as well as CRP did not have anypotential to predict refractory ischemic symptoms (R=0.043 and R=−0.034;N.S) (FIG. 2A: for quartile distribution, see Table 4B).

TABLE 2B CRP and sCD40L quartile levels at baseline. Quartiles CRP mg/LsCD40L ng/ml 1 <1.2 <14.2 2 >1.2 and <2.6 >14.2 and <26.4 3 >2.6 and<6.5 >26.4 and <33.7 4 >6.5 >33.7 All values are in ng/mlInflammation and Clinical Follow-Up

Correlation analysis for all chemokines with systemic inflammatoryparameters fibrinogen, IL-6, PAI-1 and ESR revealed no association,except for a weak correlation between CXCL10 and IL-6 levels (R=0.29;P=0.02, other data not shown). Importantly, the baseline upper quartilelevels of CCL5 as determined by multiplex were seen to correlate withthe need for revascularization procedures within the next 18 months(R=0.35; P=0.01). Furthermore, baseline upper quartile levels of CCL18correlated with the re-occurrence of unstable angina pectoris (UAP)during hospitalisation (R=0.36; P=0.007) as well as with the occurrenceof an acute coronary syndrome (ACS) during the 18-month period offollow-up (R=0.31; P=0.02)(FIGS. 2B-D). Baseline levels of sCD40L andCRP did not correlate with any of the follow-up parameters (data notshown).

PBMC Chemokine and Chemokine Receptor Expression Analysis

While the interaction of CCL5 with CCR1, CCR3, CCR4 and CCR5 is welldescribed, the actual receptor for CCL18 is as yet unknown, which makesCCL18 currently an orphan ligand¹⁷. However, CCL18 has been reported tobe a competitive inhibitor of CCL11 (eotaxin) binding to CCR3¹⁸.Therefore, we examined mRNA expression of chemokine receptors CCR1,CCR3, CCR4 and CCR5 as well as that of CCL5 and CCL18 in PBMCs, Weobserved a remarkable highly significant down-regulation of all fourinvolved chemokine receptors at baseline (t=0) compared to PBMCs att=180 (FIG. 3B). A similar temporal pattern was seen for CCL5 and CCL18,with CCL5 being abundantly expressed in PBMCs and CCL18 at only minorlevels (FIG. 3A). Subsequent FACS analysis to detect CCR3 and CCR5expression on CD3⁺ T-cells and CD14⁺ monocytes to our surprise revealeda significant elevated protein expression of CCR3 and CCR5 in both CD3⁺and CD 14⁺ cells at t=0 compared with t=180 (FIG. 4A-D) (see commentp.28). Triple staining for CD3 or CD14 with CCR3 and CCR5 showed anincreased chemokine receptor expression in the CD3⁺ population (3.1%triple positive cells at t=0 vs. 2.3% at t=180; P=0.007) and even moreprominently so in the CD14⁺ cells (32.1% vs. 5.1% at t=0 and t=180,respectively; P<0.001). An identical pattern was seen for the percentageof CCR3⁺ and CCR5⁺ cells as well as of the combined CCR3⁺/CCR5⁺ cells inthe total PBMC population (FIG. 4G-I).

To assess whether the reduced gene expression pattern at baseline werecaused by transient shifts in the leukocyte distribution profile we havemonitored the total percentage of CD14⁺ (monocytes) and CD3+ cells(T-lymphocytes) in the PBMCs. Monocyte counts were not different betweenthe two time points, whereas CD3⁺ cells were slightly decreased at t=0(54.2 vs. 66.6%; P=0.01)(FIG. 8A) see p.28. A further study revealed nodifferences in the expression ratio of CCR2:CX3CR1, a measure ofmonocyte subset distribution¹⁹, in PBMCs as well. We did however observesignificantly elevated expression levels of HNP-3, a selectiveneutrophil marker, at t=0, pointing to an enhanced release ofneutrophils during UAP (FIG. 8B) see p.28. Conceivably, the observedchanges in chemokine receptor expression at t=0 may at least partly beattributed to the increased neutrophil counts. In contrast to chemokineplasma levels, no differences in expression level were seen forchemokine receptors between stabilised and refractory patients at t=0(data not shown).

PBMC Stimulation Assay

In part however, the chemokine receptor down-regulation may reflect afeedback response on the immunomodulator burst after UAP. To verify ifthe observed expressional regulation of CCR1, CCR3, CCR4 and CCR5 inPBMCs is related to the elevated CCL5 and CCL18 levels during ischemicevents, we stimulated PBMCs with rCCL5 and/or sCCL18. After 6 hours ofstimulation, we observed no differential effect on CCR1, CCR4 and CCR5mRNA expression. In sharp contrast however, sCCL18 caused a dramaticdown-regulation in CCR3 expression, and this effect was furtheramplified by co-incubation with rCCL5 (P<0.01, FIG. 5A-D). Therefore,the down-regulation of CCR3 mRNA in PBMCs observed in vivo could becaused by the increased levels of CCL18. The down-regulation of CCR1,CCR4 and CCR5 in vivo might well be regulated by ligands other than CCL5and CCL18.

Discussion

To our knowledge, this is the first study to describe the profiling ofan extensive panel of chemokines by multiplex assay in plasma of UAPpatients in a prospective manner. Of all chemokines tested, only CCL5and CCL18 levels were, independent of other inflammatory markers andsCD40L, seen to be transiently elevated in refractory versus stabilisedpatients at baseline and to decline within 6 months after onset of theUAP symptoms. These phenomena were accompanied by a sharp, probablyCCL18 induced, decrease in mRNA expression of the cognate chemokinereceptors CCR3 and CCR5 in PBMCs at day 0 versus day 180. ConcomitantlyCCR3 and CCR5 surface expression was found to be increased at baseline,possibly reflecting a rapid receptor exposure by PBMCs during ischemicsymptoms. Both CCL5 and CCL18 also show predictive features regardingclinical outcome.

The multiplex panel contained various chemokines, which have previouslybeen linked with atherosclerosis or cardiovascular disease, such asCCL2, CCL5, CCL11, CXCL8 and CXCL10⁵. CCL5 and CCL18 were the only twochemokines that were differentially regulated at baseline betweenrefractory and stabilised patients. Refractory patients had severesustained ischemic complaints despite anti-anginal medication warrantingcoronary angiography with or without percutaneous coronary intervention.Therefore, while the levels of other chemokines that have beenimplicated in CVD were relatively unaltered and while refractorypatients do not generally differ from stabilised in the extent ofgeneral systemic inflammation, CCL5 and CCL18 might be exclusivechemokine markers of ischemia severity in patients with UAP.

CCL5 and CCL18 were selected for further temporal analysis for a 180days follow up. As previously mentioned, the role of CCL5 as aninflammatory mediator in cardiovascular disease is widely recognized,and CCL5 levels were indeed seen to be raised in patients with acutecoronary syndromes^(9,20). However, these studies examined CCL5 levelsat hospitalisation and, with one single exception, did not include aprospective study design. Only Nomura et al. showed a drop in CCL5levels 30 days in UAP patients after PCI, to levels comparable with the180 day levels in our study⁹. Our data extend this observation, as theydemonstrate that the decline in CCL5 levels is not a consequence of PCI,but an intrinsic feature of stabilised UAP patients. Although data onCCL5 reference levels are still lacking, CCL5 at 2 and 180 days postinclusion was very comparable to values reported in healthy controls byParissis et al., suggesting that CCL5 levels had returned to baselinewithin 2 days after onset of the ischemic symptoms²¹.

To gain further insight on the contribution of activated platelets tothe CCL5 peak levels, we performed a temporal assessment of sCD40L²². Weobserved significantly elevated levels of sCD40L at baseline, which isin concordance with earlier studies and reflective of the enhancedplatelet activation status in UAP^(23,24). However, the observedprogressive decline in sCD40L levels at t=2 and t=180 after UAP hasnever been documented in patients with UAP and may illustrate the rapidrestoration of sCD40L homeostasis after UAP. Furthermore, t=0 and t=2levels correlated with CCL5 levels, suggesting that activated plateletsmay, directly or indirectly, be a major source of CCL5. Apart from itsmassive secretion by activated platelets, elevated CCL5 levels duringUAP could also arise from activated T-lymphocytes and as a result ofaltered homeostasis in the ischemic tissue distal to theocclusion^(25,26). Since Rothenbacher et al. observed reduced CCL5levels in patients with stable coronary heart disease compared tocontrols, acute inflammation per se can unlikely be held responsible forthe transient increase in CCL5 during UAP²⁷. This is underscored by ourfindings, as we observed a down-regulation of CCL5 mRNA expression inPBMCs at baseline compared to 180 days after onset of the ischemia.Whether the increased response in refractory patients reflects a moreextensive platelet (or T-cell) activation or a higher capacity ofplatelets and T-cells to elaborate CCL5 remains to be determined.

Interestingly, CCL18 has not yet been associated with cardiovasculardisorders in patient cohorts. CCL18 is present at high levels in bloodand it is produced by antigen presenting cells and by eosinophils. It isthought to act in the primary immune response functioning as anattractant for T-cells, B lymphocytes and monocytes¹⁷. As previouslymentioned however, its receptor has not been identified, albeit thatCCL18 was reported to function as a neutral CCR3 antagonist¹⁸. Evidenceon a direct role of CCL18 in cardiovascular disease is not conclusiveand is limited to two descriptive studies documenting CCL18 expressionin atherosclerotic plaques and in particular at sites of reducedstability^(28,29) We now show that CCL18 plasma levels are increased inUAP patients and even more so in patients with refractory symptoms.CCL18 elevation is sustained transient but levels are lowered after 180days. The actual source of the persistent CCL18 increase after UAP isless clear. CCL18 expression was down-regulated in PBMCs at baseline,disqualifying abundant production by these cells as major source ofplasma CCL18. Conceivably, plasma levels may reflect a release fromCCL18 containing vulnerable plaques²⁸. CCL18 levels were negativelycorrelated with sCD40L levels, possibly pointing to a negative feedbackresponse upon platelet activation. Further research will have to clarifyits role in acute coronary syndromes.

It has been suggested that several chemokines can act in thepathogenesis of non-infarcted ischemic cardiomyopathy, as the prevailingreactive oxygen generation and hypoxia in the ischemic tissue willinduce a chemokine response³⁰. Illustratively, MCP-1 was seen to beup-regulated in the myocardium at least 7 days after ischemia in miceand associated with interstitial fibrosis and left ventriculardysfunction in absence of myocardial infarction⁶. CCL18 levels persistedat a high level for at least two days as well, and given its capacity toactivate fibroblasts and increase collagen production, it is tempting topropose a similar role of CCL18 in injury healing³¹. It may not onlymodulate the attraction of leukocyte subsets but, as shown by Wimmer etal., CCL18 may also play a facilatory role in bone-marrow haematopoieticstem cell function³². Therefore, elevated CCL18 levels could contributein the inflammatory response but also in progenitor cell mobilisationtowards areas of myocardial ischemia in anticipation of the myocardialrepair process.

To further stress the role of CCL5 and CCL 18 in the pathophysiology ofmyocardial ischemia, we observed a significant increase in surfaceexposure of CCR3 and CCR5 by CD3⁺ T-cells and CD14⁺ monocytes and aparadoxical mRNA down-regulation of CCR1, CCR3, CCR4 and CCR5 atbaseline. This is an intriguing and counter-intuitive observation,albeit that we are not the first to observe such a discrepancy betweenprotein and mRNA chemokine receptor expression in PBMCs from UAPpatients. In fact Damas et al. have reported a similar but oppositeeffect for CXCR4, i.e. down-regulation at the protein but up-regulationat the mRNA level in UAP compared with healthy control subjects, whilelevels of its ligand CXCL12 were lowered in patients with UAP comparedto controls³³. The rapid increase in surface protein exposure may resultfrom acute mobilisation of intracellular receptors in response toenhanced plasma levels of the cognate ligands or of other actors thatare released in unstable angina. The relative mRNA down-regulation ofchemokine receptors in PBMCs may partly reflect a shifted leukocyteprofile in UAP with a rapid mobilisation of HNP-3+ neutrophils as judgedfrom the enhanced HNP-3 expression in PBMC mRNA at t=0³⁴, and a minordecrease in CD3+ cells, while total CD14+ levels remained unaffected.Partly however it may also be attributable to a negative feedbackresponse to normalize exposed receptor levels as appears from our invitro CCL18 regulation studies (FIG. 5). The transcriptional feedbackmay be effected in direct response to exposure of the surface receptorsto CCL18, as CCR3 mRNA levels were dramatically decreased after exposureto sCCL18, thus identifying a new modulatory role of CCL18 in cardiacischemia.

Examination of CCL5 and CCL18 quartile distribution shows a clear-cutrelation with the occurrence of refractory symptoms. Furthermore, upperquartile levels also correlated with future cardiovascular events andrevascularisation procedures, whereas sCD40L and CRP, which have beenshown to have strong prognostic power in other studies³⁵⁻³⁷, did not atthis cohort size. Given the major cellular sources of CCL5 and CCL18,activated platelets and ischemic tissue, the increased levels inrefractory UAP may reflect a more pronounced thrombosis and ischemiarelated induction in these patients. Whether or not it is causal in therefractory disease progression still remains to be clarified. Regardingthe prognostic capacities of CCL5 and CCL18, the sensitivity andspecificity of the upper quartile levels of the chemokines separatelydid not exceed 80%. Combining the upper two quartiles of both chemokinesyielded a viable specificity of 90.3%, which thereby quite effectivelyrules out refractory symptoms for low CCL5 and CCL18 levels. However,although CCL5 and CCL18 may have potential as independent prospectivebiomarkers for disease, the correlations we observed between thesechemokines and clinical severity of the symptoms as well as variousfollow-up parameters, albeit very significant, are currently not strongenough on its own. Therefore, the determination of plasma CCL5 and CCL18levels, in combination with other clinical diagnostic parameters, couldadd prognostic features to the evaluation of patients with UAP. Thisissue needs to be addressed in future larger scale studies.

A few issues and limitations of this study should be noted. First, ourset up principally precluded studying control levels of these chemokinesbefore UAP. Nevertheless we believe that, as prospective analysis wereperformed in the same patients, conclusions on the temporal profile ofCCL5 and CCL18 are justified. As all patients are largely symptom freeat 180 days post UAP, we may safely assume that the latter values willapproach the pre UAP levels of CAD patients. Second, it has recentlybeen shown that statins can influence chemokine serum levels as well aschemokine receptor expression on PBMCs^(8,38). As we were in thefortunate circumstance that cohort sampling had taken place when statintherapy just began to emerge, only 8.2% of the patients of our cohortwas on statin therapy. Since our data were corrected for this minorstatin use, we believe that our results are not biased by statintherapy. Finally, the multiplex panel also comprised chemokines whichhave previously been linked to atherosclerosis or myocardial ischemia,including CCL2, CCL3, CXCL8 and CXCL10^(21,39,40). In our study,refractory unstable angina patients did not show significant differencesfor these chemokines nor for the other immunomodulators that had beenassayed. These cytokines have thus not been selected for furthertemporal analysis but we cannot a priori rule out that these cytokinesmay affect unstable angina pectoris and myocardial ischemia.

Furthermore, preliminary data in atherosclerosis prone ApoE−/− mice thatalready had developed collar induced carotid artery plaques showed thata 3 week intraperitoneal administration regimen of recombinant CCL18aggravated lesion progression by a significant 50% (FIG. 9), suggestingthat CCL18 may not only be a promising marker of cardiovascular diseasebut also a valid candidate for therapeutic intervention incardiovascular disease.

To conclude, we identified CCL5 and particularly CCL18 as relevantchemokines in UAP. Whether they play a causative role in thepathogenesis or are more indirectly involved via other mechanisms, ifthese markers harbour any further diagnostic potential and if they aresuitable therapeutic targets, needs to be addressed in future studies.

REFERENCES

-   1. Bertrand M E, Simoons M L, Fox K A, Wallentin L C, Hamm C W,    McFadden E, De Feyter P J, Specchia G, Ruzyllo W. Management of    acute coronary syndromes in patients presenting without persistent    ST-segment elevation. Eur Heart J. 2002; 23:1809-40.-   2. Hansson G K. Inflammation, Atherosclerosis, and Coronary Artery    Disease. N Engl J Med. 2005; 352:1685-1695.-   3. Charo I F, Taubman M B. Chemokines in the pathogenesis of    vascular disease. Circ Res. 2004; 95:858-66.-   4. Weber C. Platelets and chemokines in atherosclerosis: partners in    crime. Circ Res. 2005; 96:612-6.-   5. Kraaijeveld A O, de Jager S C, van Berkel T J, Biessen E A,    Jukema J W. Chemokines and Atherosclerotic Plaque Progression:    Towards Therapeutic Targeting? Curr Pharm Des. 2007; 13:1039-1052.-   6. Dewald O, Frangogiannis N G, Zoerlein M, Duerr G D, Klemm C,    Knuefermann P, Taffet G, Michael L H, Crapo J D, Welz A, Entman M L.    Development of murine ischemic cardiomyopathy is associated with a    transient inflammatory reaction and depends on reactive oxygen    species. Proc Natl Acad Sci USA. 2003; 100:2700-5.-   7. Charo I F, Ransohoff R M. The many roles of chemokines and    chemokine receptors in inflammation. N Engl J Med. 2006; 354:610-21.-   8. Damas J K, Boullier A, Waehre T, Smith C, Sandberg W J, Green S,    Aukrust P, Quehenberger O. Expression of fractalkine (CX3CL1) and    its receptor, CX3CR1, is elevated in coronary artery disease and is    reduced during statin therapy. Arterioscler Thromb Vasc Biol. 2005;    25:2567-72.-   9. Nomura S, Uehata S, Saito S, Ostumi K, Ozeki Y, Kimura Y. Enzyme    immunoassay detection of platelet-derived microparticles and RANTES    in acute coronary syndrome. Thromb Haemost. 2003; 89:506-12.-   10. McDermott D H, Yang Q, Kathiresan S, Cupples L A, Massaro J M,    Keaney J F, Jr., Larson M G, Vasan R S, Hirschhorn J N, O'Donnell C    J, Murphy P M, Benjamin E J. CCL2 polymorphisms are associated with    serum monocyte chemoattractant protein-1 levels and myocardial    infarction in the Framingham Heart Study. Circulation. 2005;    112:1113-20.-   11. de Lemos J A, Morrow D A, Sabatine M S, Murphy S A, Gibson C M,    Antman E M, McCabe C H, Cannon C P, Braunwald E. Association between    plasma levels of monocyte chemoattractant protein-1 and long-term    clinical outcomes in patients with acute coronary syndromes.    Circulation. 2003; 107:690-5.-   12. de Jager W, to Velthuis H, Prakken B J, Kuis W, Rijkers G T.    Simultaneous detection of 15 human cytokines in a single sample of    stimulated peripheral blood mononuclear cells. Clin Diagn Lab    Immunol. 2003; 10:133-9.-   13. Verheggen P W, de Maat M P, Cats V M, Haverkate F, Zwinderman A    H, Kluft C, Bruschke A V. Inflammatory status as a main determinant    of outcome in patients with unstable angina, independent of    coagulation activation and endothelial cell function. Eur Heart J.    1999; 20:567-74.-   14. de Jager W, Prakken B J, Bijlsma J W, Kuis W, Rijkers G T.    Improved multiplex immunoassay performance in human plasma and    synovial fluid following removal of interfering heterophilic    antibodies. J Immunol Methods. 2005.-   15. Cusack M R, Marber M S, Lambiase P D, Bucknall C A, Redwood S R.    Systemic inflammation in unstable angina is the result of myocardial    necrosis. J Am Coll Cardiol. 2002; 39:1917-23.-   16. Kennon S, Price C P, Mills P G, Ranjadayalan K, Cooper J, Clarke    H, Timmis A D. The effect of aspirin on C-reactive protein as a    marker of risk in unstable angina. J Am Coll Cardiol. 2001;    37:1266-70.-   17. Schutyser E, Richmond A, Van Damme J. Involvement of CC    chemokine ligand 18 (CCL18) in normal and pathological processes. J    Leukoc Biol. 2005; 78:14-26.-   18. Nibbs R J, Salcedo T W, Campbell J D, Yao X T, Li Y, Nardelli B,    Olsen H S, Morris T S, Proudfoot A E, Patel V P, Graham G J. C-C    chemokine receptor 3 antagonism by the beta-chemokine macrophage    inflammatory protein 4, a property strongly enhanced by an    amino-terminal alanine-methionine swap. J Immunol. 2000;    164:1488-97.-   19. Tacke F, Alvarez D, Kaplan T J, Jakubzick C, Spanbroek R, Llodra    J, Garin A, Liu J, Mack M, van Rooijen N, Lira S A, Habenicht A J,    Randolph G J. Monocyte subsets differentially employ CCR2, CCR5, and    CX3CR1 to accumulate within atherosclerotic plaques. J Clin Invest.    2007; 117:185-94.-   20. Steppich B A, Moog P, Matissek C, Wisniowski N, Kuhle J,    Joghetaei N, Neumann F J, Schomig A, Ott I. Cytokine profiles and T    cell function in acute coronary syndromes. Atherosclerosis. 2007;    190:443-51.-   21. Parissis J T, Adamopoulos S, Venetsanou K F, Mentzikof D G,    Karas S M, Kremastinos D T. Serum profiles of C-C chemokines in    acute myocardial infarction: possible implication in postinfarction    left ventricular remodeling. J Interferon Cytokine Res. 2002;    22:223-9.-   22. Andre P, Nannizzi-Alaimo L, Prasad S K, Phillips D R.    Platelet-derived CD40L: the switch-hitting player of cardiovascular    disease. Circulation. 2002; 106:896-9.-   23. Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A,    Solum N O, Forfang K, Froland S S, Gullestad L. Enhanced levels of    soluble and membrane-bound CD40 ligand in patients with unstable    angina. Possible reflection of T lymphocyte and platelet involvement    in the pathogenesis of acute coronary syndromes. Circulation. 1999;    100:614-20.-   24. Garlichs C D, Eskafi S, Raaz D, Schmidt A, Ludwig J, Herrmann M,    Klinghammer L, Daniel W G, Schmeisser A. Patients with acute    coronary syndromes express enhanced CD40 ligand/CD154 on platelets.    Heart. 2001; 86:649-55.-   25. Nelson P J, Kim H T, Manning W C, Goralski T J, Krensky A M.    Genomic organization and transcriptional regulation of the RANTES    chemokine gene. J Immunol. 1993; 151:2601-12.-   26. Weber C, Schober A, Zernecke A. Chemokines: key regulators of    mononuclear cell recruitment in atherosclerotic vascular disease.    Arterioscler Thromb Vasc Biol. 2004; 24:1997-2008.-   27. Rothenbacher D, Muller-Scholze S, Herder C, Koenig W, Kolb H.    Differential expression of chemokines, risk of stable coronary heart    disease, and correlation with established cardiovascular risk    markers. Arterioscler Thromb Vasc Biol. 2006; 26:194-9.-   28. Reape T J, Rayner K, Manning C D, Gee A N, Barnette M S, Burnand    K G, Groot P H. Expression and cellular localization of the CC    chemokines PARC and ELC in human atherosclerotic plaques. Am J    Pathol. 1999; 154:365-74.-   29. Papaspyridonos M, Smith A, Burnand K G, Taylor P, Padayachee S,    Suckling K E, James C H, Greaves D R, Patel L. Novel candidate genes    in unstable areas of human atherosclerotic plaques. Arterioscler    Thromb Vasc Biol. 2006; 26:1837-44.-   30. Frangogiannis N G, Entman M L. Chemokines in myocardial    ischemia. Trends Cardiovasc Med. 2005; 15:163-9.-   31. Atamas S P, Luzina I G, Choi J, Tsymbalyuk N, Carbonetti N H,    Singh I S, Trojanowska M, Jimenez S A, White B. Pulmonary and    activation-regulated chemokine stimulates collagen production in    lung fibroblasts. Am J Respir Cell Mol Biol. 2003; 29:743-9.-   32. Wimmer A, Khaldoyanidi S K, Judex M, Serobyan N, Discipio R G,    Schraufstatter I U. CCL18/PARC stimulates hematopoiesis in long-term    bone marrow cultures indirectly through its effect on monocytes.    Blood. 2006; 108:3722-9.-   33. Damas J K, Waehre T, Yndestad A, Ueland T, Muller F, Eiken H G,    Holm A M, Halvorsen B, Froland S S, Gullestad L, Aukrust P. Stromal    cell-derived factor-1 alpha in unstable angina: potential    antiinflammatory and matrix-stabilizing effects. Circulation. 2002;    106:36-42.-   34. Hansen P R. Role of neutrophils in myocardial ischemia and    reperfusion. Circulation. 1995; 91:1872-85.-   35. Kinlay S, Schwartz G G, Olsson A G, Rifai N, Sasiela W J, Szarek    M, Ganz P, Libby P. Effect of atorvastatin on risk of recurrent    cardiovascular events after an acute coronary syndrome associated    with high soluble CD40 ligand in the Myocardial Ischemia Reduction    with Aggressive Cholesterol Lowering (MIRACL) Study. Circulation.    2004; 110:386-91.-   36. Varo N, de Lemos J A, Libby P, Morrow D A, Murphy S A, Nuzzo R,    Gibson C M, Cannon C P, Braunwald E, Schonbeck U. Soluble CD40L:    risk prediction after acute coronary syndromes. Circulation. 2003;    108:1049-52.-   37. Lindahl B, Toss H, Siegbahn A, Venge P, Wallentin L. Markers of    myocardial damage and inflammation in relation to long-term    mortality in unstable coronary artery disease. FRISC Study Group.    Fragmin during Instability in Coronary Artery Disease. N Engl J Med.    2000; 343:1139-47.-   38. Veillard N R, Braunersreuther V, Arnaud C, Burger F, Pelli G,    Steffens S, Mach F. Simvastatin modulates chemokine and chemokine    receptor expression by geranylgeranyl isoprenoid pathway in human    endothelial cells and macrophages. Atherosclerosis. 2006; 188:51-8.-   39. Gerszten R E, Garcia-Zepeda E A, Lim Y C, Yoshida M, Ding H A,    Gimbrone M A, Jr., Luster A D, Luscinskas F W, Rosenzweig A. MCP-1    and IL-8 trigger firm adhesion of monocytes to vascular endothelium    under flow conditions. Nature. 1999; 398:718-23.-   40. Heller E A, Liu E, Tager A M, Yuan Q, Lin A Y, Ahluwalia N,    Jones K, Koehn S L, Lok V M, Aikawa E, Moore K J, Luster A D,    Gerszten R E. Chemokine CXCL10 promotes atherogenesis by modulating    the local balance of effector and regulatory T cells. Circulation.    2006; 113:2301-12.

EXAMPLE 2 CCL3 (MIP-1α) levels are elevated during acute coronarysyndromes and show strong prognostic power for future ischemic events.

Methods

Patient Cohorts

Mission

Study populations were compiled from the MISSION! intervention study¹².The AMI patient group consisted of 44 patients (54.5% male; mean age61.8±11.6 years) diagnosed with AMI on the basis of ECG and clinicalchemical parameters (elevated troponin and creatine kinase levels). Thecontrol group represented 22 non-symptomatic age and sex matchedsubjects (54.5% male; mean age 61.7±12.8), not suffering from manifestcoronary artery disease (Table 5). Baseline blood samples of AMIpatients were taken within 2 hours after hospitalization and within 6hours upon onset of AMI. Patients suffering from autoimmune disease,malignancies, chronic inflammatory diseases as rheumatoid arthritis orreceiving immunosuppressant or chemotherapy were excluded from thestudy. This study was approved by the local ethics committee and allpatients and healthy volunteers gave informed consent before beingrecruited. The investigation conformed to the principles outlined in theHelsinki Declaration.

Aprais

Plasma samples of patients with unstable angina, derived from the welldefined APRAIS (Acute Phase Reaction and Ischemic Syndromes) study, wereused to determine circulating CCL3 levels¹³. In brief, 54 patients whowere admitted to the emergency department of the Leiden UniversityMedical Center between March and September 1995 with unstable anginapectoris Braunwald class IIIB were included and followed for up to 18months. Venous blood samples were obtained on admission (t=0) after 2(t=2) and 180 days after admission (t=180), centrifuged and plasmaaliquots were stored at −80° C. until further analysis. All patients hadreceived standard medical therapy, i.e. aspirin 300 mg orally,nitro-glycerine intravenously and heparin infusion based titrated to theactivated partial thromboplastin time. All subjects gave writteninformed consent and the study protocol was approved by the EthicsCommittee of the Leiden University Medical Center.

Multiplex Chemokine Assay

Circulating chemokines levels of CCL2, CCL3, CCL5, CCL11, CCL17, CCL18,CCL22, CXCL8, CXCL9, and CXCL10 as well as four reference cytokines weredetermined in the MISSION! cohort, as well as CCL3 levels in the APRAIScohort, by using a highly sensitive fluorescent microsphere basedreadout as described earlier^(14,15). Briefly, plasma samples werefiltered and subsequently diluted with 10% normal rat and mouse serum(Rockland, Gilvertsville, Pa.) to block residual non-specific antibodybinding. 1000 microspheres were added per chemokine (10 μl/well) in atotal volume of 60 μl, together with standard and blank samples, and thesuspension incubated for 1 hour in a 96 well filter plate at roomtemperature (RT). Then, 10 μl of biotinylated antibody mix (16.5 μg/ml)was added and incubated for 1 hour at RT. After washing with PBS-1%BSA-0.5% Tween 20, beads were incubated with 50 ng/well streptavidinR-phycoerythrin (BD Biosciences, San Diego, Calif.) for 10 minutes.Finally, beads were washed again with PBS-1% BSA-0.5% Tween 20, and thefluorescence intensity was measured in a final volume of 100 μlhigh-performance ELISA buffer (Sanquin, Amsterdam, the Netherlands).Measurements and data analysis were performed with the Bio-PlexSuspension Array system in combination with the Bio-Plex Managersoftware version 3.0 (Bio-Rad laboratories, Hercules, Calif.).

Murine Myocardial Infarction

Mice were anaesthetized and artificially ventilated with a mixture ofoxygen and N₂O [1:2 (vol/vol)] using a rodent ventilator (HarvardApparatus, Holliston, Mass.) to which 2-2.5% isoflurane (AbbottLaboratories, Hoofddorp, the Netherlands) was added for anesthesia.Myocardial infarction was induced by permanent ligation of the proximalleft anterior descending coronary artery with a sterile 7/0 silk suture(Ethicon, Johnson & Johnson, Amersfoort, the Netherlands). Three hoursafter ligation the mice were sacrificed, PBMCs and spleens were isolatedfor flow cytometric analysis and plasma was harvested for chemokinedetection. All animal procedures were approved by the Animal EthicsCommittee of Leiden University.

Elisa And Other Assays

Human as well as murine CCL3 levels (Biosource, Carlsbad, Calif.),murine CXCL10 (R&D systems, Minneapolis, Minn.) and murine IL-6(eBioscience, San Diego, Calif.) were determined by sandwich Elisaassays as described by the manufacturers protocol. Baseline inflammatoryparameters in the APRAIS cohort, such as C-reactive protein, fibrinogenand erythrocyte sedimentation rate (ESR), were determined as describedpreviously¹³. Soluble CD40 ligand (sCD40L) was determined via a highlysensitive immunoassay (Quantakine HS, R&D Systems, Minneapolis, Minn.).

Flow Cytometry

PBMCs were isolated from whole blood by ablation of the erythrocytes.Splenocytes were isolated by squeezing spleens through a 70 μm cellstrainer (BD falcon, BD Biosciences, San Jose, Calif.). After collectiontotal blood cells and splenocytes were incubated with erythrocytes lysisbuffer for 5 minutes on ice. Cells were centrifuged for 5 minutes andresuspended in lysis buffer. Residual erythrocytes were lysed by 5minute incubation on ice. Cells were washed twice with PBS and counted.Consequently cells were stained for CD4, CCR3, CCR5 (BD Biosciences),CD8, F4/80 (eBioscience) and CXCR3 (US biological, Swampscott, Mass.)surface markers by adding 0.25 μg antibody per sample. After 45 minutesincubation on ice, cells were washed with PBS and subsequently analyzedby flow cytometry (FACScalibur, BD biosciences).

Statistical Methods

Statistical analysis was performed using SPSS version 13.0 (SPSS,Chicago, Ill.) All values are expressed as mean E standard error ofmean. Differences in risk factor distribution between the control andthe AMI group were analyzed with a Fishers Exact probability test.Chemokine data were tested for normal distribution by use of aKolmogorov-Smirnov analysis. Non-Gaussian distributed data were analyzedby a Mann-Whitney U test, whereas normally distributed variables wereanalyzed by Student's t-test. Correlation analysis with inflammatoryparameters was performed by Spearman's rank correlation test. Covariateadjustment for risk factors was performed by a univariate linearregression test. Quartile distribution of CCL3 was assessed and used forChi-Square testing to associate elevated levels of CCL3 with futurecardiovascular events. A P-value <0.05 was considered significant.

Results

Mission Patient Statistics

Two sub-cohorts were compiled at a 2:1 ratio as a pilot study revealedthat the standard deviation in cytokine levels in the AMI population wason average 1.5 fold higher than that of the control subjects. AMI andcontrol sub-cohorts were matched for gender, age and risk factors knownto be associated with inflammatory status (type 2 diabetes mellitus,hypertension and hyperlipidemia). The AMI cohort encompassed a higherfraction of smokers and ex-smokers than the control cohort (56.8% in AMIcompared to 22.7% in controls; P=0.01; Table 5). Therefore, allchemokine values were adjusted for smoking by univariate analysis. Allproteins were all well within detectable range of the used assay.

TABLE 5 MISSION! Patient Characteristics Acute Myocardial P- ControlsInfarction value Age (years) 61.7 ± 2.6 61.8 ± 1.8 0.96 Male/Female12/10 24/20 1.00 Diabetes Mellitus 3 (13.6%)  6 (13.6%) 1.00Hypertension 8 (36.3%) 11 (25%) 0.39 Total Cholesterol  5.6 ± 0.3 mmol/L 6.0 ± 0.1 mmol/L 0.14 Smoking 5 (22.7%) 25 (56.8%)*; 0.01 4 (18.1%)ex-smokers  4 (9.1%) ex-smokersReference Panel

As a control for the validity of the multiplex assay a panel ofreference cytokines and cell adhesion markers was included in theanalysis. In compliance with previous findings plasma levels of IL-2(0.07±0.06 pg/ml in controls vs. 0.65±0.28 in AMI; P=0.003), TNF-α(1.05±0.32 pg/ml in controls vs. 2.4±0.72 in AMI; P=0.03), sICAM-1(476.1±80.7 ng/ml in controls vs. 713.0±49.9 in AMI; P=0.04) and IL-6(9.8±4.1 in controls compared to 23.7±8.0 pg/ml in AMI; P=0.04) weresignificantly elevated in AMI patients (Table 6). Other generalinflammation markers as IL-1α, IFN-γ and sVCAM-1 remained unchanged(data not shown), thereby showing that the AMI patient cohort was notenriched in subjects with a general hyperinflammatory status.

TABLE 6 APRAIS CCL3 t = 0 quartile levels as determined by multiplexQuartiles CCL3 (pg/ml) 1 <41 2 >41 and <53 3 >53 and <83 4 >83Chemokines

Plasma levels of the CC chemokines CCL3 (39.8 pg/ml, 21.3-50.3 IQR incontrols compared to 47.8 pg/ml, 39.6-67.2 IQR in AMI; P=0.01: FIG. 13A)and CCL5 (13.4 ng/ml, 6.4-29.2 IQR in controls compared to 33.3 ng/ml,19.1-45.3 in AMI; P=0.001: FIG. 14B) were significantly up-regulated inAMI compared to control patients (Table 7). After correction forcardiovascular risk factors CCL3 and CCL5 remained significantlyelevated during AMI (P=0.025 and P=0.006 respectively). Of the CXCchemokines only CXCL8 (4.2±0.50 pg/ml in controls compared to 6.8±0.56in AMI; P=0.01; FIG. 13C) was significantly up-regulated, while CXCL10(255.1±47.2 pg/ml in control vs. 162.6±20.3 in AMI; P=0.002: FIG. 13D)was down-regulated in AMI compared to controls. After covariateadjustment both CXCL8 and CXCL10 remained significantly changed (P=0.02and P=0.04 respectively). All other measured chemokines were notdifferentially regulated during AMI (Table 7).

TABLE 7 Mean Cytokine and Chemokine values Control AMI P P* IL-2 0.07 ±0.06 pg/ml 0.65 ± 0.28 pg/ml ↑ 0.003 0.047 IL-6 9.8 ± 4.1 pg/ml 23.8 ±8.0 pg/ml ↑ 0.04 0.07 TNFα 0.6 pg/ml, (0-1.6) 1.4 pg/ml, (0.5-2.4) ↑0.03 0.01 sICAM-1 476 ± 80.7 ng/ml 714 ± 50.0 ng/ml ↑ 0.045 <0.001 CCL2305 ± 81 pg/ml 522 ± 77 pg/ml = 0.08 0.14 CCL3 49.8 pg/ml (21.3-50.6)47.7 pg/ml, (39.6-67.2) ↑ 0.02 0.025 CCL5 13.4 ng/ml (6.4-29.2) 33.3ng/ml, (19.8-45.3) ↑ 0.001 0.006 CCL11 15.9 pg/ml, (12.7-22.0) 21.2pg/ml, (13.6-29.8) = 0.27 0.33 CCL17 16.4 pg/ml, (10.5-21.4) 16.6 pg/ml,(8.6-28.9) = 0.46 0.26 CCL18 555 ± 186 ng/ml 681 ± 160 ng/ml = 0.18 0.85CCL22 356 pg/ml, (264-409) 371 pg/ml, (296-549) = 0.11 0.08 CXCL8 3.5pg/ml, (1.9-4.3) 5.1 pg/ml, (3.5-7.4) ↑ 0.004 0.02 CXCL9 163 ± 51 pg/ml155 ± 25 pg/ml = 0.16 0.87 CXCL10 255 ± 47.4 pg/ml 120 ± 20.3 pg/ml ↓0.001 0.004Reference (IL-2, IL-6, TNF-α and sICAM-1) and chemokine panel ofmeasured parameters containing P value and corrected P value (P*) afteradjustment for smoking. Values are expressed as mean±SEM or median withIQR when appropriate.Aprais

To verify this observation we compared CCL3 levels of the MISSION!cohort with those of the APRAIS cohort as described earlier refer toexample 1. Inter-study analysis showed that patients with UAP alsodisplayed similar increased CCL3 plasma levels compared to the MISSION!AMI patients (FIG. 11A). Next, we performed a temporal analysis ofcirculating CCL3 levels in the APRAIS cohort of patients with unstableangina pectoris. Plasma samples from baseline (t=0), t=2 and t=180, asanalyzed by ELISA, revealed a significant decrease of CCL3 levels att=180 compared with t=0 as well as t=2 (t=0 7.57 pg/ml; t=2 6.49 pg/ml;t=180 4.31 pg/ml, P<0.001) (FIG. 11B). Although absolute CCL3 plasmalevels detected by ELISA were lower, comparison of both techniquesrevealed a highly significant correlation (R=0.92, P<0.001). Next, wesought to assess if CCL3 plasma levels had any potential to predictclinical outcome. Given the cohort size, multiplex CCL3 t=0 plasmalevels were therefore categorized into quartiles and analyzed forcorrelation with the occurrence of ischemic symptoms during orimmediately after hospitalisation and/or acute coronary syndromes (forquartile distribution, see Table 6). Upper quartile levels of CCL3 werehighly predictive for the occurrence of acute coronary syndromes duringfollow-up (Likelihood ratio 11.52; P<0.01) and recurrent unstable anginapectoris during hospitalisation (Likelihood ratio 14.63; P<0.01) (FIG.12A,B). Cardiac death during follow-up also showed a significantassociation, although less strong (Likelihood ratio 7.92; P<0.05)(datanot shown). Finally, CCL3 did not correlate with any of the inflammatoryparameters (data not shown). However, sCD40L levels revealed asignificant negative correlation with CCL3 levels (R=−0.44; P=0.001),suggestive of a feedback response upon platelet activation.

Unlike CCL5 and CCL18, CCL3 levels was not predictive of a refractorynature of UAP (early stage) but highly significantly so of more mid termevents occurring within 180 days after UAP.

Murine Myocardial Infarction

The obtained results in humans suggest an important role for CCL3 inischemic myocardial injury. To ascertain whether the enhanced chemokineswere ischemia related we performed myocardial infarction experiments inmice. Since the chemokines CCL5 and CXCL8 have been extensively studiedregarding atherothrombosis and AMI we turned our interest to CCL3 andCXCL10. To induce acute myocardial infarction the left anteriordescending coronary artery was ligated in C57B16 mice. CCL3 levels were,in concurrence with the earlier MISSION! findings, significantlyelevated after AMI (33.2±1.5 vs. 76.4±37.4 pg/ml in ligated animals;P=0.02) (FIG. 14B). As a control for the AMI model, levels of theischemia related cytokine IL-6 were measured^(16,17). IL-6 levels weresignificantly up-regulated after ligation (0.67±0.26 in sham vs.1.34±0.46 ng/ml in ligated animals; P=0.007, FIG. 14A). Surprisingly thelevels of CXCL10 were, opposed to the MISSION! findings, significantlyenhanced after AMI (157.3±64.8 in sham operated compared to 310.6±86.6pg/ml in ligated animals; P=0.03) (FIG. 14C). In addition PBMCs wereharvested and analyzed for chemokine receptor expression on differentcell subsets. As expected, the total T-cell population was enhanced inthe circulation after ligation (14.1±3.8% in controls vs. 32.8±14.4% inligated mice; P=0.038) while no effects were seen on splenic T-cells(P=0.9, FIGS. 15A and D respectively). Moreover the number of bothcirculating as well as splenic macrophages was not regulated by ischemicinjury (data not shown). More extensive analysis of the T-cellpopulation revealed a specific enrichment of CCR5⁺ T-cells (8.0±2.0% incontrols compared to 11.4±1.4% in ligated animals; P=0.02) (FIG. 15B).The enrichment in circulatory CCR5⁺ T-cells was accompanied by areduction in splenic CCR5⁺ T-cells (19.95±0.5% vs. 14.1±3.1%; P=0.004)(FIG. 15E). CCR3 is the known receptor for the CCL3 related chemokineCCL4. As CCL3 and CCL4 are usually co-regulated we also analyzed PBMCsand splenocytes for CCR3 expression. The numbers of circulating CCR3⁺T-cells was very low. Analysis showed a slight, albeit not significant(P=0.24), increase in circulating CCR3⁺ T-cells (data not shown). Nodifferences in splenic CCR3⁺ T-cells were evident (data not shown).Taken together these data suggest a CCL3 specific migration of T-cellsfrom the secondary lymphoid organs towards the site of ischemic injury.In addition expression of the CXC chemokine receptor CXCR3 wasdetermined on the circulating T-cells as well. In concurrence with theenhanced CXCL10 levels, the number of circulating CXCR3⁺ T-cells wassignificantly increased after LAD ligation (29.1±1.9% vs. 43.5±5.7%;P=0.04) (FIG. 15C). However no effects on CXCR3⁺ splenic T-cells wereapparent (P=0.78) (FIG. 15F).

Preliminary data suggest that CCL3 levels not only are predictive of therisk of future cardiovascular events, but may also be causallyimplicated in disease development as atherosclerotic plaque growth inthe aortic sinus of hyperlipidemic LDL receptor knockout mice with aleukocyte deficiency in CCL3 is significantly lower (−60%) than that inmice with normal leukocyte production of CCL3 (FIG. 10).

REFERENCES

-   1. Hansson G K. Inflammation, atherosclerosis, and coronary artery    disease. N Engl J Med. 2005; 352:1685-95.-   2. Laing K J, Secombes C J. Chemokines. Dev Comp Immunol. 2004;    28:443-60.-   3. Weber C. Novel mechanistic concepts for the control of leukocyte    transmigration: specialization of integrins, chemokines, and    junctional molecules. J Mol Med. 2003; 81:4-19.-   4. Weber C, Schober A, Zernecke A. Chemokines: key regulators of    mononuclear cell recruitment in atherosclerotic vascular disease.    Arterioscler Thromb Vasc Biol. 2004; 24:1997-2008.-   5. Olson T S, Ley K. Chemokines and chemokine receptors in leukocyte    trafficking. Am J Physiol Regul Integr Comp Physiol. 2002; 283    :R7-28.-   6. Kraaijeveld A O, de Jager S C, van Berkel T J, Biessen E A,    Jukema J W. Chemokines and atherosclerotic plaque progression:    towards therapeutic targeting? Curr Pharm Des. 2007; 13:1039-52.-   7. Mause S F, von Hundelshausen P, Zernecke A, Koenen R R, Weber C.    Platelet Microparticles. A Transcellular Delivery System for    RANTES-Promoting Monocyte Recruitment on Endothelium. Arterioscler    Thromb Vasc Biol. 2005.-   8. Simeoni E, Winkelmann B R, Hoffmann M M, Fleury S, Ruiz J,    Kappenberger L, Marz W, Vassalli G. Association of RANTES G-403A    gene polymorphism with increased risk of coronary arteriosclerosis.    Eur Heart J. 2004; 25:1438-46.-   9. Boger C A, Fischereder M, Deinzer M, Aslanidis C, Schmitz G,    Stubanus M, Banas B, Kruger B, Riegger G A, Kramer B K. RANTES gene    polymorphisms predict all-cause and cardiac mortality in type 2    diabetes mellitus hemodialysis patients. Atherosclerosis. 2005.-   10. Frangogiannis N G. The role of the chemokines in myocardial    ischemia and reperfusion. Curr Vasc Pharmacol. 2004; 2:163-74.-   11. Tarzami S T, Miao W, Mani K, Lopez L, Factor S M, Berman J W,    Kitsis R N. Opposing effects mediated by the chemokine receptor    CXCR2 on myocardial ischemia-reperfusion injury: recruitment of    potentially damaging neutrophils and direct myocardial protection.    Circulation. 2003; 108:2387-92.-   12. Liem S S, van der Hoeven B L, Oemrawsingh P V, Bax J J, van der    Bom J G, Bosch J, Viergever E P, van Rees C, Padmos I, Sedney M I,    van Exel H J, Verwey H F, Atsma D E, van der Velde E T, Jukema J W,    van der Wall E E, Schalij M J. MISSION!: optimization of acute and    chronic care for patients with acute myocardial infarction. Am    Heart J. 2007; 153:14 e1-11.-   13. Verheggen P W, de Maat M P, Cats V M, Haverkate F, Zwinderman A    H, Kluft C, Bruschke A V. Inflammatory status as a main determinant    of outcome in patients with unstable angina, independent of    coagulation activation and endothelial cell function. Eur Heart J.    1999; 20:567-74.-   14. de Jager W, to Velthuis H, Prakken B J, Kuis W, Rijkers G T.    Simultaneous detection of 15 human cytokines in a single sample of    stimulated peripheral blood mononuclear cells. Clin Diagn Lab    Immunol. 2003; 10:133-9.-   15. de Jager W, Prakken B J, Bijlsma J W, Kuis W, Rijkers G T.    Improved multiplex immunoassay performance in human plasma and    synovial fluid following removal of interfering heterophilic    antibodies. J Immunol Methods. 2005; 300:124-35.-   16. LaFramboise W A, Bombach K L, Dhir R J, Muha N, Cullen R F,    Pogozelski A R, Turk D, George J D, Guthrie R D, Magovern J A.    Molecular dynamics of the compensatory response to myocardial    infarct. J Mol Cell Cardiol. 2005; 38:103-17.-   17. Shu J, Ren N, Du J B, Zhang M, Cong H L, Huang T G. Increased    levels of interleukin-6 and matrix metalloproteinase-9 are of    cardiac origin in acute coronary syndrome. Scand Cardiovasc J. 2007;    41:149-54.-   18. Miyao Y, Yasue H, Ogawa H, Misumi I, Masuda T, Sakamoto T,    Morita E. Elevated plasma interleukin-6 levels in patients with    acute myocardial infarction. Am Heart J. 1993; 126:1299-304.-   19. Mizia-Stec K, Gasior Z, Zahorska-Markiewicz B, Janowska J, Szulc    A, Jastrzebska-Maj E, Kobielusz-Gembala I. Serum tumour necrosis    factor-alpha, interleukin-2 and interleukin-10 activation in stable    angina and acute coronary syndromes. Coron Artery Dis. 2003;    14:431-8.-   20. Wang Y N, Che S M, Ma A Q. Clinical significance of serum    cytokines IL-1beta, sIL-2R, IL-6, TNF-alpha, and IFN-v in acute    coronary syndrome. Chin Med Sci J. 2004; 19:120-4.-   21. de Lemos J A, Hennekens C H, Ridker P M. Plasma concentration of    soluble vascular cell adhesion molecule-1 and subsequent    cardiovascular risk. J Am Coll Cardiol. 2000; 36:423-6.-   22. Zhou R H, Shi Q, Gao H Q, Shen B J. Changes in serum    interleukin-8 and interleukin-12 levels in patients with ischemic    heart disease in a Chinese population. J Atheroscler Thromb. 2001;    8:30-2.-   23. Hashmi S, Zeng Q T. Role of interleukin-17 and    interleukin-17-induced cytokines interleukin-6 and interleukin-8 in    unstable coronary artery disease. Coron Artery Dis. 2006;    17:699-706.-   24. Parissis J T, Adamopoulos S, Venetsanou K F, Mentzikof D G,    Karas S M, Kremastinos D T. Serum profiles of C-C chemokines in    acute myocardial infarction: possible implication in postinfarction    left ventricular remodeling. J Interferon Cytokine Res. 2002;    22:223-9.-   25. Nomura S, Uehata S, Saito S, Osumi K, Ozeki Y, Kimura Y. Enzyme    immunoassay detection of platelet-derived microparticles and RANTES    in acute coronary syndrome. Thromb Haemost. 2003; 89:506-12.-   26. Mause S F, von Hundelshausen P, Zernecke A, Koenen R R, Weber C.    Platelet microparticles: a transcellular delivery system for RANTES    promoting monocyte recruitment on endothelium. Arterioscler Thromb    Vasc Biol. 2005; 25:1512-8.-   27. Guan E, Wang J, Norcross M A. Identification of human macrophage    inflammatory proteins 1alpha and 1beta as a native secreted    heterodimer. J Biol Chem. 2001; 276:12404-9.-   28. Vandervelde S, van Luyn M J, Rozenbaum M H, Petersen A H, Tio R    A, Harmsen M C. Stem cell-related cardiac gene expression early    after murine myocardial infarction. Cardiovasc Res. 2007; 73:783-93.-   29. Hou Y, Plett P A, Ingram D A, Rajashekhar G, Orschell C M, Yoder    M C, March K L, Clauss M. Endothelial-monocyte-activating    polypeptide II induces migration of endothelial progenitor cells via    the chemokine receptor CXCR3. Exp Hematol. 2006; 34:1125-32.-   30. Waeckel L, Mallat Z, Potteaux S, Combadiere C, Clergue M, Duriez    M, Bao L, Gerard C, Rollins B J, Tedgui A, Levy B I, Silvestre J S.    Impairment in postischemic neovascularization in mice lacking the    CXC chemokine receptor 3. Circ Res. 2005; 96:576-82.    Materials and Methods    Animals

LDLr^(−/−) mice were obtained from the local animal breeding facility.Mice were maintained on sterilized regular chow (RM3; Special DietServices, Essex, U.K.). Drinking water was supplied with antibiotics (83mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sucroseand was provided ad libitum. Animal experiments were performed at theanimal facilities of the Gorlaeus laboratories of the Leiden University.All experimental protocols were approved by the ethics committee foranimal experiments of Leiden University.

Temporal Expression Profile

Male LDLr^(−/−) mice were fed a Western type diet containing 0.25%cholesterol and 15% cacaobutter (Special Diet Services, Sussex, UK) twoweeks prior to surgery and throughout the experiment. To determine geneexpression levels in (n=20) mouse plaques, atherosclerotic carotidartery lesions were induced by perivascular collar placement asdescribed previously¹. Mice were anaesthetized by subcutaneous injectionof ketamine (60 mg/kg, Eurovet Animal Health, Bladel, The Netherlands),fentanyl citrate and fluanisone (1.26 mg/kg and 2 mg/kg respectively,Janssen Animal Health, Sauderton, UK). From 0 to 8 weeks after collarplacement every two weeks a subset of 4 mice was sacrificed. The animalswere anaesthetized as described above and perfused through the leftcardiac ventricle with PBS and exsanguinated by femoral arterytranssection. Subsequently, both common carotid arteries were removedand snap-frozen in liquid nitrogen for optimal RNA preservation. Thespecimens were stored at −80° C. until further use.

RNA Isolation

Two or three carotids were pooled per sample and homogenized bygrounding in liquid nitrogen with a pestle. Total RNA was extracted fromthe tissue using Trizol reagent according to manufacturer's instructions(Invitrogen, Breda, The Netherlands). RNA was reverse transcribed usingM-MuLV reverse transcriptase (RevertAid, MBI Fermentas, Leon-Roth) andused for quantitative analysis of gene expression with an ABI PRISM 7700Taqman apparatus (Applied Biosystems, Foster City, Calif.) as describedpreviously², using murine hypoxanthine phosphoribosyltransferase (HPRT)and cyclophilin A (CypA) as standard housekeeping genes (Table 8).

Bone Marrow Transplantation

To induce bone marrow aplasia, male LDLr^(−/−) recipient mice wereexposed to a single dose of 9 Gy (0.19 Gy/min, 200 kV, 4 mA) total bodyirradiation using an Andrex Smart 225 Röntgen source (YXLONInternational) with a 6-mm aluminum filter 1 day before transplantation.Bone marrow was isolated from male CCL3^(−/−) or littermates by flushingthe femurs and tibias. Irradiated recipients received 0.5×10⁷ bonemarrow cells by tail vein injection and were allowed to recover for 6weeks. Animals were placed on a western type diet containing 0.25%cholesterol and 15% cacao butter (SDS) diet for 12 weeks andsubsequently sacrificed. Twenty four hours prior to sacrifice a subsetof animals were injected intraperitoneally with lipopolysaccharide (LPS)(Salmonella minnesota R595 (Re) (List Biological Laboratories Inc.,Campbell, Calif.)). Plasma levels of CCL3 were determined by sandwichElisa(Biosource, Carlsbad, Calif., according to the manufacturer'sprotocol) to confirm impaired CCL3 production from leukocytes.

Histological Analysis

Cryostat sections of the aortic root (10 μm) were collected and stainedwith Oil-red-O. Lesion size was determined in 5 sections of the aorticvalve leaflet area. Corresponding sections on separate slides werestained immunohistochemically with an antibody directed against amacrophage specific antigen (MOMA-2, monoclonal rat IgG2b, dilution1:50; Serotec, Oxford, UK). Goat anti-rat IgG-AP (dilution 1:100; Sigma,St. Louis, Mo.) was used as secondary antibody and NBT-BCIP (Dako,Glostrup, Denmark) as enzyme substrates. Masson's trichrome staining(Sigma, St. Louis, Mo.) was used to visualize collagen (blue staining).Neutrophils were visualized by Naphtol AS-D Chloroacetate Esterase stainaccording to the manufacturer's protocol (Sigma).

Macrophage Stimulation

Serum deprived RAW264.7 macrophages were stimulated with 10 μg/ml ox-LDLor 1 ng/ml LPS for 24 hours. Total RNA was isolated for real time PCR toassess CCL3 expression. Serum deprived RAW 264.7 macrophages werestimulated with recombinant CCL3 (10 or 100 ng/ml) for 24 hours.Subsequently [³H]-Thimidine (1 μCi/well, specific activity 24 Ci/mmol;Amersham Biosciences, The Netherlands) was added to each well and cellswere allowed to proliferate for another 24 hours. Cells were rinsedtwice with cold PBS and subsequently lysed with 0.1M NaOH. The amount of[³H]-thymidine incorporation was measured using a liquid scintillationanalyzer (Tri-Carb 2900R).

Cyclophosphamide Induced Neutropenia

Female CCL3^(−/−) mice or WT control received an intraperitoneal (i.p)injection of cyclophosphamide (6 mg/mouse) to deplete blood neutrophilsas described previously^(3, 4). Blood samples were taken via the tailvein regularly and blood cell differentiation was determined on a Sysmexcell differentiation apparatus (Goffin Meyvis, Etten-Leur, Nederland).

In Vivo Chemotaxis

Female CCL3−/− mice or WT control received an i.p. injection of 500 ngrecombinant KC (Peprotech, Rocky Hill, N.J.) or PBS. Two hours laterblood and peritoneal cells were isolated and analyzed for neutrophilcomposition by flow cytometry.

Flow Cytometry

Peritoneal leukocytes were harvested by peritoneal cavity lavage withPBS. Crude peripheral blood mononuclear cells (PBMC) and peritonealleukocytes were incubated at 4° C. with erythrocyte lysis buffer (155 mMNH₄CL in 10 mM Tris/HCL, pH 7.2) for 5 minutes. Cells were centrifugedfor 5 minutes at 1500 rpm, resuspended in lysis buffer to removeresidual erythrocytes. Cells were washed twice with PBS. Cellsuspensions were incubated with 1% normal mouse serum in PBS and stainedfor the surface markers CD11b, GR1 and CD71 (eBioscience, San Diego,Calif.) at a concentration of 0.25 μg Ab/200,000 cells. Subsequentlycells were subjected to flow cytrometric analysis (FACSCalibur, BDBiosciences, San Diego, Calif.). FACS data were analyzed with CELLQuestsoftware (BD Biosciences).

Statistical Analysis

Data are expressed as mean±SEM. A 2-tailed Student's t-test was used tocompare individual groups, while multiple groups were compared with aone-way ANOVA and a subsequent Student-Newman-Keuls multiple comparisonstest. Non-parametric data were analyzed using a Mann-Whitney U test. Alevel of P<0.05 was considered significant.

Results

Temporal expression analysis of atherosclerotic lesions in LDLr−/− miceshowed a clearcut, transient upregulation of CCL3 in initial plaques (2weeks after collar placement). At more advanced stages of lesionprogression CCL3 is returning to its original level. This expression isinitially accomponied by increased expression of macrophage marker CD68of which its levels remain high at later time points. The expression ofCD36 is somewhat delayed as compared to CD68 and CCL3 (FIG. 16). Theexpression profiles suggest that CCL3 may be involved in the criticalrecruitment of inflammatory cells to atherosclerotic lesion sites. Invitro exposure of RAW 264.7 macrophages to ox-LDL leads to a moderateinduction of CCL3 expression, while the TLR4 ligand LPS strongly inducesMIP1α at mRNA level (FIG. 17).

To assess effects of hematopoietic CCL3 deficiency on leukocytemigration and activation as well as on atherogenesis we reconstitutedLDLr−/− mice with CCL3−/− bone marrow. CCL3 deficiency did not influencebody weight or total cholesterol levels during the course of theexperiment (data not shown). Plasma MIP1α levels were not significantlydifferent between CCL3−/− chimeras and littermate controls (2.4±0.8pg/ml in WT vs. 0.9±0.6 pg/ml in CCL3^(−/−) chimeras; p=0.1, FIG. 17C).The CCL3 deficient phenotype was much more pronounced after in vivotreatment with LPS. Circulating MIP1α levels 24 h after LPS treatmentwere robustly increased in WT but not in CCL3−/− chimeras (14.7±0.4pg/ml in control compared to 2.1±1.0 pg/ml in CCL3^(−/−) chimeras;p=0.00005, FIG. 18A).

Lesion development in the aortic root of CCL3−/− chimeras was reduced bya significant 31% (135.1±76.5×10³ μm² in CCL3^(−/−) compared to198.4±51.4×10³ μm² in controls; P=0.04, FIG. 19A). The percentage ofintimal MoMa-2⁺ macrophages was not different between groups (19.3±2.6%in controls vs. 22.9±3.0% in CCL3^(−/−), FIG. 18B), suggesting that CCL3alone may not be very critical in macrophage accumulation andproliferation in the atherosclerotic plaque. CD3 T cell numbers were notinfluenced by CCL3 deficiency (2.9±1.2 T cells/mm² plaque in controlsand 2.6±1.5 T cells/mm² plaque in CCL3−/−, FIG. 18D). In contrast, theamount of plaque neutrophils (7.0±0.7 in WT compared to 2.9±0.8/mm²intimal tissue in CCL3^(−/−) plaques; p=0.001, FIG. 18E), as well asneutrophil adherence were significantly reduced in CCL3^(−/−) plaques(FIG. 18F). As measure of lesion progression stage intimal collagendeposition was determined. The percentage of collagen in CCL3^(−/−)plaques was not influenced by CCL3 deficiency (7.5±1.4 in WT compared to5.7±1.0% in CCL3^(−/−) chimeras, FIG. 18C).

CCL3 deficiency did not influence the total number of circulating whiteblood cells in WT and CCL3^(−/−) transplanted animals (4.4±0.7 in WT vs.3.9±0.6×10⁶ cells/ml in CCL3^(−/−), FIG. 19A) and the number ofcirculating monocytes was not affected by CCL3 deficiency as well(7.7±1.1 in WT vs. 8.9±1.0% in CCL3^(−/−) chimeras, FIG. 19B).Interestingly the percentage of circulating neutrophils wassignificantly decreased in CCL3^(−/−) chimeras (35.3±3.9 in WT vs.23.6±2.5% in CCL3^(−/−) chimeras; p=0.02, FIG. 19C).

The decreased neutrophil numbers may result from a reduced half life oran impaired differentiation and stromal egress of neutrophils. Toinvestigate this, animals were treated with a single injection ofcyclophosphamide and the neutrophil elimination/repopulation kineticswas monitored for 10 days. Basal white blood cell number and cellularcomposition was not different between WT controls and CCL3^(−/−) mice.CCL3 deficient cells were slightly more sensitive to cyclophosphamidetreatment (FIG. 20A,B) as white blood cell half life was significantlyenhanced in CCL3^(−/−) mice compared to WT (1.09±0.07 days in WTcompared to 0.89±0.06 days in CCL3^(−/−); p=0.04, FIG. 20C) and appearedequally distributed over the neutrophil and lymphocyte subset (FIG.20C). Thus CCL3 deficient mice show a decreased neutrophil half lifewhich concurs with the reduced numbers of circulating and plaqueneutrophils in this strain. Repopulation of cells initiated 5 days postinjection and was similar between CCL3^(−/−) and WT controls (FIG. 20D)

Next we assessed the chemotactic response of WT and CCL3^(−/−)neutrophils towards a gradient of the major chemokine in neutrophilrecruitment, KC. Two hours after i.p. injection of KC, WBCs andperitoneal leukocytes were isolated and analyzed for neutrophil content.Circulating neutrophil numbers were similar between WT and CCL3^(−/−)animals (6.1±1.0 in WT compared to 5.3±1.0 in CCL3^(−/−), FIG. 21A).Surprisingly, given the reduced circulating neutrophil numbers,CCL3^(−/−) animals had slightly enhanced neutrophil numbers in theperitoneum under normal conditions (0.6±0.5% in WT compared to 1.4±0.07,p=0.2, data not shown). KC injections robustly induced neutrophilmigration towards the peritoneum of control animals. Peritonealneutrophil counts after KC injections in CCL3^(−/−) animals were onlymarginally lower compared to WT animals (12.3±0.4 in controls comparedto 10.2±1.9 in CCL3^(−/−) animals, data not shown). However theinduction of neutrophil influx was decreased in CCL3^(−/−) animals (20×induction in WT compared to 7.5× induction in CCL3^(−/−), p=0.003; FIG.21C), suggestive of impaired chemotaxis of CCL3^(−/−) neutrophils underconditions of inflammation.

Interestingly plaque formation was attenuated as a result of leukocytespecific absence of CCL3, which may be due to a decreased accumulationof neutrophils in the plaque. Collectively our data indicate thatdeficiency of CCL3 will translate in a reduced neutrophil half life andto a impaired CXCR2 dependent accumulation of neutrophils in the plaque,which subsequently will translate into attenuated plaque progression.

Discussion

Chemokine mediated migration of leukocytes into the vessel wall is anessential step in atherosclerotic lesion formation and progression⁵. TheCC chemokine CCL3 can interact with chemokine receptors CCR4, CCR1 andCCR5, of which the latter two have been implicated in atherogenesis.Combined with the upregulated aortic expression during atherogenesis⁶,and its potent chemotactic effect on T cells, macrophages andneutrophilsTNF-α⁷, a role of this chemokine in atherogenesis isconceivable. Here we show that leukocytes are the prime source of CCL3under conditions of inflammation and that leukocyte CCL3 deficiencyattenuates plaque development by altering neutrophil half life andreducing neutrophil accumulation.

In vitro experiments clearly established that activated macrophages area rich source of CCL3, which is in concurrence with earlier data⁸.Moreover baseline levels of CCL3 in the circulation were seen to be onlypartly of leukocyte origin but almost exclusively produced by leukocytesduring LPS elicited inflammatory responses^(2,9). Expression profiles ofatherosclerotic lesion development revealed that CCL3 is mainlyupregulated during early lesion progression, suggesting that CCL3 isinvolved in plaque inflammation⁶. Atherogenesis in CCL3^(−/−) mice wassignificantly attenuated, but no effects on macrophage or T cell contentwere apparent. Interestingly, hematopoietic and systemic deficiency ofone of the CCL3 receptors, CCR1, led to acceleratedatherosclerosis^(10, 11). CCR1 deficient plaques contained moremacrophages and T cells and CCR1^(−/−) T cells produced more IFNγ¹⁰.Conversely functional deficiency of CCR5, either in the hematopoieticlineage or systemically, was shown to reduce atherosclerotic lesiondevelopment and plaques contained less macrophages and T cells^(11, 12).Antagonism of CCR5 by use of Met-RANTES similarly attenuatedatherosclerosis development, macrophage and T cell content. FurthermoreMet-Rantes treatment resulted in lower expression levels of CCR5, butnot of its ligand CCL3¹³. CCL3 was shown to have a higher bindingaffinity for CCR5^(14, 15), suggestive that CCR5 mediated effects areprimary during a chronic low rate inflammation, while acute substantialinflammation might correct these effects via CCR1 signalling. Thephenotypic change seen in hematopoietic CCL3 deficiency seems to be moreconsistent with that of impaired CCR5 function, albeit that we did notsee any noticeable effects on plaque macrophage content. This indicatesthat, although CCL3 might influence inflammatory cell migration, it isnot crucial in monocyte or T cell migration towards the plaque.

Neutrophils were, until recently, not implicated in the pathogenesis ofatherosclerosis. However more and more data are accumulating thatsupport an active role of this subset of white blood cells in thisdisease. Naruka et al showed plaque neutrophil infiltrates to beassociated with acute coronary events¹⁶. Experimental support came fromvan Leeuwen and coworkers showing the abundant presence of neutrophilsin advanced mouse plaques¹⁷, and from a collaborative expansion afterblockage of CXCR4¹⁸. Plaque neutrophils are potent inflammatory cellsacting in a narrow time span. Neutrophils are associated with increasedintimal apoptosis and a pro-inflammatory phenotype¹⁸. Conceivablyneutrophil accumulation in atherosclerotic lesions can induce plaquedestabilization as a result of enhanced inflammation, necrotic coreformation as a consequence of oxidative injury and matrix degradation byrelease of neutrophil elastases. CCL3 has been reported to be able toaugment neutrophil chemotaxis induced by the pro-inflammatory cytokineTNFα in a CCR5 dependent manner⁷. In concurrence with these findings weshow attenuated neutrophil migration to and diapedesis into the plaquein hematopoietic CCL3 deficiency. Moreover in vivo neutrophil migrationtowards KC (murine IL8 analogue) was reduced in CCL3^(−/−) mice. Thisindicates that IL-8, similar to TNFα, can induce CCL3 mediatedneutrophil migration.

Another intriguing option is that CCL3 affects neutrophil homeostasis.During inflammation, circulating neutrophil numbers were significantlylower in CCL3^(−/−) mice, which fits well with the notion that apoptosisof neutrophils is regarded as a protective measure to dampen acuteinflammatory responses and prevent unwanted tissue damage¹⁹. Terminallymatured neutrophils therefore show a sharply reduced half life.Moreover, they have impaired migration and degranulation^(20, 21). Weobserved a clear effect of CCL3−/− on neutrophil elimination kinetics,as the half life of CCL3 deficient neutrophils was decreased. Howeverrepopulation of neutrophils was not influenced by CCL3 deficiency,showing that neutrophil maturation and stromal release per se are notinfluenced. These data suggest that CCL3^(−/−) neutrophils are moresensitive to cyclophosphamide, and perhaps other pro-apoptotic signalsleading to a reduced half life.

Taken together our data clearly establish a causal role for neutrophilsin the development of atherosclerosis. Furthermore we hypothesize thatunder conditions of inflammation leukocyte derived CCL3 can, possibly inconcert with TNFα, alter neutrophil homeostasis and enhance neutrophilchemotaxis towards the atherosclerotic plaque to accelerate lesionformation.

TABLE 8 RT-PCR primer sequences and sources. Gene forward primer (5′-3′)reverse primer (5′-3′) CCL3 GCCACATCGAGGGACTCTTC GATGGGGGTTGAGGAACGTG ACD36 GTTCTTCCAGCCAATGCCTT ATGTCTAGCACACCATAAGAT T GTA CAGTT CD68CCTCCACCCTCGCCTAGTC TTGGGTATAGGATTCGGATTT GA HPRT TTGCTCGAGATGTCATGAAGAGCAGGTCAGCAAAGAACTTA GA TAG CypA CCATTTCAAGAAGCAGCGTTATTTTGTCTTAACTGGTGGGT T CTGT

REFERENCES

-   1. von der Thusen J H, van Berkel T J, Biessen E A. Induction of    rapid atherogenesis by perivascular carotid collar placement in    apolipoprotein E-deficient and low-density lipoprotein    receptor-deficient mice. Circulation. Feb. 27, 2001;    103(8):1164-1170.-   2. Kasama T, Strieter R M, Standiford T J, et al. Expression and    regulation of human neutrophil-derived macrophage inflammatory    protein 1 alpha. J Exp Med. Jul. 1, 1993; 178(1):63-72.-   3. Zuluaga A F, Salazar B E, Rodriguez C A, et al. Neutropenia    induced in outbred mice by a simplified low-dose cyclophosphamide    regimen: characterization and applicability to diverse experimental    models of infectious diseases. BMC Infect Dis. 2006; 6:55.-   4. Spellberg B J, Collins M, French S W, et al. A phagocytic cell    line markedly improves survival of infected neutropenic mice. J    Leukoc Biol. August 2005; 78(2):338-344.-   5. Charo I F, Taubman M B. Chemokines in the pathogenesis of    vascular disease. Circ Res. Oct. 29, 2004; 95(9):858-866.-   6. Moos M P, John N, Grabner R, et al. The lamina adventitia is the    major site of immune cell accumulation in standard chow-fed    apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol.    November 2005; 25(11):2386-2391.-   7. Montecucco F, Steffens S, Burger F, et al. Tumor necrosis    factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1)    up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on    human neutrophils through defined signalling pathways. Cell Signal.    March 2008; 20(3):557-568.-   8. Fahey T J, 3rd, Tracey K J, Tekamp-Olson P, et al. Macrophage    inflammatory protein 1 modulates macrophage function. J Immunol. May    1, 1992; 148(9):2764-2769.-   9. Harrison L M, van den Hoogen C, van Haaften W C, et al. Chemokine    expression in the monocytic cell line THP-1 in response to purified    shiga toxin 1 and/or lipopolysaccharides. Infect Immun. January    2005; 73(1):403-412.-   10. Potteaux S, Combadiere C, Esposito B, et al. Chemokine receptor    CCR1 disruption in bone marrow cells enhances atherosclerotic lesion    development and inflammation in mice. Mol Med. January-December    2005; 11(1-12):16-20.-   11. Potteaux S, Combadiere C, Esposito B, et al. Role of bone    marrow-derived CC-chemokine receptor 5 in the development of    atherosclerosis of low-density lipoprotein receptor knockout mice.    Arterioscler Thromb Vasc Biol. August 2006; 26(8):1858-1863.-   12. Braunersreuther V, Zernecke A, Arnaud C, et al. Ccr5 but not    Ccr1 deficiency reduces development of diet-induced atherosclerosis    in mice. Arterioscler Thromb Vasc Biol. February 2007;    27(2):373-379.-   13. Veillard N R, Kwak B, Pelli G, et al. Antagonism of RANTES    receptors reduces atherosclerotic plaque formation in mice. Circ    Res. Feb. 6, 2004; 94(2):253-261.-   14. Neote K, DiGregorio D, Mak J Y, et al. Molecular cloning,    functional expression, and signaling characteristics of a C-C    chemokine receptor. Cell. Feb. 12, 1993; 72(3):415-425.-   15. Samson M, Labbe O, Mollereau C, et al. Molecular cloning and    functional expression of a new human CC-chemokine receptor gene.    Biochemistry. Mar. 19, 1996; 35(11):3362-3367.-   16. Naruko T, Ueda M, Haze K, et al. Neutrophil infiltration of    culprit lesions in acute coronary syndromes. Circulation. Dec. 3,    2002; 106(23):2894-2900.-   17. van Leeuwen M, Gijbels M J, Duijvestijn A, et al. Accumulation    of myeloperoxidase-positive neutrophils in atherosclerotic lesions    in LDLR−/− mice. Arterioscler Thromb Vasc Biol. January 2008;    28(1):84-89.18-   18. Zernecke A, Bot I, Djalali-Talab Y, et al. Protective role of    CXC receptor 4/CXC ligand 12 unveils the importance of neutrophils    in atherosclerosis. Circ Res. Feb. 1, 2008; 102(2):209-217.-   19. Luo H R, Loison F. Constitutive neutrophil apoptosis: mechanisms    and regulation. Am J Hematol. April 2008; 83(4):288-295.-   20. Savill J. Apoptosis in resolution of inflammation. J Leukoc    Biol. April 1997; 61(4):375-380.-   21. Whyte M K, Meagher L C, MacDermot J, et al. Impairment of    function in aging neutrophils is associated with apoptosis. J    Immunol. Jun. 1, 1993; 150(11):5124-5134.

The invention claimed is:
 1. A method for identifying a subject atincreased risk of a future acute cardiovascular syndrome or event, themethod comprising: a) obtaining a sample from a subject; b) determiningan amount of at least one compound selected from the group consisting ofCCL3 (chemokine (C-C motif) ligand 3), CCL18 (chemokine (C-C motif)ligand 18), and CCL5 (chemokine (C-C motif) ligand 5) by contacting thesample with: i) an antibody specific for CCL3, and quantifying theamount of CCL3 bound to the antibody, ii) an antibody specific forCCL18, and quantifying the amount of CCU18 bound to the antibody, oriii) an antibody specific for CCL5, and quantifying the amount of CCL5bound to the antibody; c) identifying a subject as having an increasedrisk of a future acute cardiovascular syndrome or event if the samplefrom the subject was determined to have greater than 41 pg/ml CCL3,greater than 40.3 ng/ml CCL5, or greater than 39.3 ng/ml CCL18; and d)performing follow-up on the subject identified with an increased riskusing standard medical therapy.
 2. The method according to claim 1,wherein the cardiovascular syndrome or event may comprise coronaryartery disease, atherosclerosis, acute myocardial infarction,arteriosclerosis, unstable angina pectoris, embolism, deep veinthrombosis, stroke, congestive heart failure or arrhythmia.
 3. Themethod according to claim 1, wherein the indication of an increased riskof a future acute cardiovascular syndrome or event may be used formonitoring the status and/or progression of said syndrome or event. 4.The method according to claim 1, wherein the indication of an increasedrisk of a future acute cardiovascular syndrome or event may be used formonitoring therapeutic regimes and/or clinical trials in order to detectwhether or not a particular treatment may be effective in reducing anincreased risk of an acute cardiovascular syndrome or event.
 5. Themethod according to claim 1, wherein the sample is a cell taken from thesubject or a sample of a body fluid of the subject, which may be derivedfrom blood or from a blood fraction.
 6. The method according to claim 1,wherein the amount of compound bound to an antibody is detected by amethod selected from the group consisting of an immunoassay, an enzymelinked immunoassays (ELISA), a fluorescence based assays, a dissociationenhanced lanthanide fluoroimmunoassay (DELFIA), a radiometric assays, amultiplex immunoassays, and a cytrometric bead assays (CBA).
 7. Themethod according to claim 1, further comprising assessing clinicalsymptoms, determining the level of at least one other compound in thesubject, or a combination thereof, wherein the at least one othercompound is a biomarker indicative of cardiovascular disease or apredisposition thereto.
 8. The method according to claim 7, wherein theat least one other compound is selected from the group consisting ofCXSCL 10 (IP-10), C-Reactive Protein, troponin I, creatine kinase,creatine kinase MB, CD40L, high density lipoprotein (HDL), myoglobin andinterleukin-6.
 9. A method for identifying a subject at increased riskof a future acute cardiovascular syndrome or event, which methodcomprises: a) determining a level of at least one compound selected fromthe group consisting of CCL3, CCL18, and CCL5, in a sample from asubject to be tested using a method selected from the group consistingof an immunoassay, an enzyme linked immunoassays (ELISA), a fluorescencebased assays, a dissociation enhanced lanthanide fluoroimmunoassay(DELFIA), a radiometric assays, a multiplex immunoassays and acytrometric bead assays (CBA); b) identifying a subject as having anincreased risk of a future acute cardiovascular syndrome or event if thesample from the subject was determined to have greater than 41 pg/mlCCL3, greater than 40.3 ng/ml CCL5, or greater than 39.3 ng/ml CCL18;and c) performing follow-up on the subject identified with an increasedrisk using standard medical therapy.
 10. A method for identifying asubject at increased risk of a future acute cardiovascular syndrome orevent, the method comprising: a) obtaining a sample from a subject; b)determining an amount of each compound selected from the groupconsisting of CCL3 (chemokine (C-C motif) ligand 3), CCL18 (chemokine(C-C motif) ligand 18), and CCL5 (chemokine (C-C motif) ligand 5) bycontacting the sample with: i) an antibody specific for CCL3, andquantifying the amount of CCL3 bound to the antibody, ii) an antibodyspecific for CCL18, and quantifying the amount of CCL18 bound to theantibody, and iii) an antibody specific for CCL5, and quantifying theamount of CCL5 bound to the antibody; c) identifying a subject as havingan increased risk of a future acute cardiovascular syndrome or event ifthe sample from the subject was determined to have greater than 41 pg/mlCCL3, greater than 40.3 ng/ml CCL5, and greater than 39.3 ng/ml CCL18;and d) performing follow-up on the subject identified with an increasedrisk using standard medical therapy.
 11. The method of claim 10, whereinthe subject is identified as having an increased risk of a future acutecardiovascular syndrome or event if the sample from the subject wasdetermined to have greater than 53 pg/ml CCL3.
 12. The method of claim10, wherein the subject is identified as having an increased risk of afuture acute cardiovascular syndrome or event if the sample from thesubject was determined to have greater than 83 pg/ml CCL3.
 13. Themethod of claim 10, wherein the subject is identified as having anincreased risk of a future acute cardiovascular syndrome or event if thesample from the subject was determined to have greater than 66 ng/mlCCL18.
 14. The method of claim 10, wherein the subject is identified ashaving an increased risk of a future acute cardiovascular syndrome orevent if the sample from the subject was determined to have greater than130 ng/ml CCL18.
 15. The method of claim 10, wherein the subject isidentified as having an increased risk of a future acute cardiovascularsyndrome or event if the sample from the subject was determined to havegreater than 53 pg/ml CCL3, greater than 40.3 CCL5, and greater than 66ng/ml CCL18.
 16. The method of claim 10, wherein the sample is selectedfrom the group consisting of blood and serum.